Overview Of Prominent Lal Tests

According to a Parenteral Drug Association (PDA) survey "Current Practices in Endotoxin and Pyrogen Testing in Biotechnology" (55), as of 1990 the gel-clot assay was the most used assay (77%) with the remainder, the kinetic chromogenic and turbidimetric LAL assays, representing less than one-third of users responding. Most of respondents (71%) indicated for the continued use of the rabbit pyrogen test for at least some final product testing; however, was very seldom indicated for any other sample tests. With that survey now over fifteen years old, current indications are that the use of the kinetic methods have ballooned while the end point, pyrogen and gel-clot tests have diminished significantly. It also seems fair to say that the recombinant factor C test will have to climb a fairly steep learning curve but that eventually it may end up the primary test. Albeit, in laboratories with fewer samples, the simplicity and elegance of the gel-clot assay may still be preferred.

Assay Characteristics of the General LAL Reaction with Endotoxin

Early on, Levin and Bang (56) described three critical properties of the gelation of LAL in the presence of LPS that formed the basis for subsequent assays, including:

1. The increase in optical density (OD) that accompanies coagulation is due to the increase of clottable protein

2. The concentration of LPS determines the rate of the OD increase

3. The reaction occurs in the shape of a sigmoid curve (i.e., a plateau, a rapid rise, and a final plateau).d

Each of these factors is demonstrated graphically (Fig. 4).

The total amount of clotted protein formed depends upon the initial LAL concentration. An excess of LAL is provided for LAL testing and the amount of clotted protein eventually ends up the same, regardless of the amount of endotoxin in the sample. The end result of the enzymatic cascade is the formation of a web of clotted protein. The gel-clot and end point tests take a single time point reading from the data to determine if the reaction reached an assigned level during the assigned time, whereas the kinetic tests are "watching" (at the appropriate wavelength) throughout the entire course of the reaction. The endotoxin concentration determines the rate of protein clot formation and thus the OD change over time as determined by measuring the time to reach an assigned mOD value. The rate of OD formation is then related to the standard curve formed using control standard endotoxin. It can be seen from a plate that sits out that all wells containing endotoxin will eventually form a dark colorimetric or turbidimetric solution regardless of the endotoxin concentration.

Besides the basic gelation of LAL in the presence of LPS, the two methods of observing the assay include the end point and kinetic assays. In the end point test, the reaction proceeds until it is stopped by the user via the addition of a stop reagent (such as acetic acid) at which point the OD readings are recorded for all sample and standard curve points. The drawbacks associated with the end point method of observing the reaction are (i) necessity of the user attention at the end of data collection (typically 30 minutes) and (ii) the limited standard curve range (a single log). In the kinetic assay, the spectrophotometer records the OD reading continuously [as often as the operator determines via the software settings, but within the manufacturer's recommendations—typically 1:30 to 2:00 minute intervals (due to the amount of internal data that can otherwise needlessly accumulate thus creating very large files to backup)]. Kinetic testing measures the rate of the OD change, by recording the time it takes to reach a preset OD setting called the "onset"

dSee Hurley's paper on methods of endotoxemia detection: Endotoxemia: methods of detection and Clinical Correlates. Clin Microbiol Rev 1995; 8(2):268 - 292.

Turbidimetric Method Lal
FIGURE 5 Time to reach mOD as a measure of the rate of change of three kinetic assays: chromogenic, turbidimetric, and silkworm plasma. Abbreviation: OD, optical density.

or "threshold" time (Fig. 5). The kinetic assay plots the log of the resulting reaction time in seconds against the log of the endotoxin concentration of the known standards and can span several logs (typically two to four) and proceeds unattended, thus overcoming the two disadvantages presented by the end point tests.

The Gel-Clot Assay

The gel-clot assay is a simple test not far removed from Levin and Bang's original observations. Until recently it has been the most widely used procedure for the detection of endotoxin in solutions. When equal parts of LAL are combined with a dilution of sample containing endotoxin, one can expect to see gelation in the amount equivalent to the endotoxin sensitivity [called lambda (A)] of the given lysate. A series of dilutions will reveal the approximate content of a sample with those samples containing equal to or greater than the given sensitivity being positive, and those below the sensitivity not clotting the mixture. The solutions are incubated at a temperature correlating to a physiological temperature (37°C) for one hour and clots are observed by inverting the tubes 180 degrees. In 10 mm x 75 mm depyrogenated test tubes, the clot must remain in the bottom of the tube when inverted. The method is considered semi-quantitative because the true result obtained (indicated by the last gelled sample in the series) is actually somewhere between the two serial dilutions. This is because the result cannot be extrapolated between the (usually two-fold) dilution tubes as it is in the kinetic and end point assays via the use of a mathematical standard curve extrapolated over the entire range of standards although one may continue to assay tighter and tighter dilutions to arrive at the approximate endotoxin concentration via the gel-clot method. But such is the labor intensity of pinpointing a more approximate result via the gel-clot assay.

Because commercial lysates are available with various standardized end points (sensitivities), the assay can be used to quantify the level of endotoxin in a particular solution or product. The level of endotoxin is calculated by multiplying the reciprocal of the highest dilution (the dilution factor) of the test solution giving a positive end point by the sensitivity of the lysate preparation. For example, if the sensitivity of the LAL employed were 0.03EU/mL and the dilution end point were 1:16, then the endotoxin concentration would be 16 x 0.03 = 0.48 EU/mL. For products administered by weight, the result in EU/mL is divided by the initial test solution potency (as reconstituted or as per the liquid in the vial) to give a result in EU/unit (EU/mg, EU/insulin unit, EU/mL of drug, etc.) that can then be compared to the tolerance limit specification. The geometric mean calculation is used for assays as opposed to the pass-fail limit test (that is reported as a "less-than" number if there is no activity).

Characteristics of Kinetic Methods

Given that kinetic assays continue to be the overwhelming area of growth in LAL testing (listed as a primary reason for the harmonization of endotoxin standards in IS-2), it is relevant to discuss the history and details of kinetic testing. The first kinetic chromogenic test was developed by Nakamura et al. (58). Nakamura et al. (58) tested eight different synthetic substrates and only one showed good reactivity (l00%) with endotoxin [BZ (a-N-benzoyl)-Ile-Glu-Gly-Arg-PNA (p-nitroanilide)]. This substrate had previously been used with factor Xa. The development of the chromogenic assay was largely driven by the desire to accurately determine the endotoxin content for bacteremia (60), endotoxemia (61), and bodily fluids such as blood plasma and cerebrospinal fluids (62).

The first kinetic test resembling today's test was developed by Ditter et al. (62). The following passage describes the early kinetic chromogenic test:

... the OD is measured every minute for 100 min to obtain the complete kinetics of each reaction in the microtiter plate in a modified photometer (Titertek Multiskan, Flow Laboratories) providing a constant temperature of 37°C. As an index for each of the 96 reactions, the maximal increase in OD per minute (Odmax/min) within 100 minutes is computed. This procedure results in a standard curve that is linear over an extremely wide range, in contrast to the limited linearity of endotoxin standard curves obtained by photometric end point methods.

Urbaschek et al. (61) included an internal standardization method that is used today, namely, the practice of including known standards contained within the sample (positive product control) as described [though Uraschek used the control standard endotoxin (CSE) in a series of concentrations in the product thus mimicking the preparation of the standard curve, a method seldom used today but described in the 1987 FDA Guideline]:

This internal standardization, based on a mathematical model, allows the quantification of the unknown endotoxin concentration in the sample and at the same time reveals the extent of sample-related interferences. Whereas an endotoxin standard curve is linear in samples not containing endotoxin, it shows a characteristic deviation of this slope when the sample contains endotoxin ... (63).

Though at the time the group could find no improved utility in this method versus the turbidimetric kinetic method for the test of bodily fluids, nevertheless, the test served as a precursor to later efforts by Lindsay et al. (57) to improve upon the kinetic methods (chromogenic and turbidimetric) as alternatives to end point tests. Current kinetic tests can be used to span up to 5 logs (i.e., 0.005 to 50 EU/ mL). Moreover, whereas early chromogenic tests employed multiple reagents (LAL, chromogenic substrate, and buffer), current tests have an increased ease of use in that the LAL, substrate, and buffer are colyophilized into a single vial.

Table 4 shows standard curve values obtained from a kinetic chromogenic assay (A = 0.05 EU/mL) tested on a commercial reader/software system. These

TABLE 4 Relative Advantages and Disadvantages of Major Limulus Amebocyte Lysate Test Types

Kinetic end point tests versus gel-clot method

Kinetic quantitative extrapolation of an unknown result between standards via linear or polynomial regression.

Less prone to variation due to user technique.

Provides "on board" documentation and calculation capabilities for consumables and products used in the test.

The mathematical treatment of data allows for the observance of trends and for the setting of numerical system suitability and assay acceptance criteria. May have different interference profiles than gel-clot assays (useful if the gel-clot assay will not give a valid result at a sensitive level). Assays may be automated.

Lambda may be varied by changing the bottom value of the standard curve (within the limits of the given LAL), thus allowing the MVD to be extended for diffifult-to-test (interfering) products. Kinetic tests versus end point tests

Quantifies a result over a range of several logs (i.e., the difference between the highest and lowest standard curve points) versus a single log. Tests to completion without user intervention after LAL addition—precision, speed, and accuracy improved.

Chromogenic versus turbidimetric tests (kinetic and end point)

Calculates a result over a range of several logs (i.e., the difference between the highest and lowest standard curve points) versus a single log. Tests to completion without user intervention after LAL addition.

Turbidity determinations are made based on the physical blocking of transmitted light (like nephlometry).

Chromogenic methods (end-point and kinetic) are not limited by particulate constraints associated with Beer's Law (absorbance is directly proportional to common parameters such as well depth). The chromogenic method may be applied to turbid samples. The turbidimetric method may be applied to samples with a yellow tint. Recombinant factor C (fluorescent test)

May provide sample suitability advantages as it does not contain unknown factors associated with the blood of the horseshoe crab (i.e., no glucan pathway). Fluorescence associated with emission not absorbance as per kinetic methods. rFC not susceptible to lot-to-lot variability as it is not a product of seasonal Limulus blood harvest and purification.

Considered an alternate assay by USP standards and requires validation of USP parameters including comparative study to accepted USP method. It is not a blood product and therefore, technically, not subject to the same constraints in its manufacture and distribution; however, the converse of this is less guidance in its use to date. Provides a much needed safeguard against catastrophic loss of Limulus.

Abbreviations: MVD, maximum valid dilution; LAL, limulus amebocyte lysate.

are the data from which the kinetic reader software uses the formulas referenced in Attachment A for the result calculations given unknown sample reaction times.

Among the most significant advantages of kinetic and end point testing over the gel-clot assay is that they allow for the quantitative extrapolation of an unknown result between standard points. (See Table 4 for a summary of the relative advantages presented by each major type of LAL test.) In the gel-clot test, results are limited by the dilutions that can be made, typically in a two-fold fashion. In this manner, the gel-clot assay can only reveal that the true value is between the positive and negative recovery in the given test tubes (i.e., the "break point"), whereas in the kinetic test samples are pipetted into a 96 well microtiter plate layered with LAL and read photometricallly by a spectrophotometer set on 405 or 340 nm (kinetic chromogenic and turbidimetric). The color or turbidity reaction that occurs between LAL and endotoxin is recorded in the form of the time in seconds that it takes a sample to reach a threshold OD reading as a defined setting in the reader's software (OD or mOD). The log of the time obtained for each sample is plotted against the log of the endotoxin content obtained in the same test for known standards.

The gel-clot quantification approach has been widely used to monitor in-process materials and water, but has been largely supplanted by kinetic tests due to the ability of kinetic assays to quantify and extrapolate accurate results over a wide range of endotoxin concentration. A positive control consisting of a product sample spiked with a known concentration of endotoxin and a negative control using nonpyrogenic water is used in LAL test procedure to ensure the lack of interference in the sample matrix. Although a simple clot end point may be adequate for routine release testing of various pharmaceuticals, the ability to quantify endotoxin is invaluable for troubleshooting production-related pyrogen problems. Daily monitoring of plant water and in-process testing can alert production personnel to potential pyrogen problems before they become critical. Corrective action can be taken to reduce pyrogen loads and levels of endotoxin at this time. Using the gel-clot assay, one would not see the increase in activity until the sample forms a clot. Thus there is little or no warning prior to failing a given lot of water sample (or anything else).

Kinetic Turbidimetric Assay

Turbidity is a precursor to gel-clot formation and, therefore, the turbidimetric test is clearly an extension of the gel-clot assay. This LAL reagent contains enough coagu-logen to form turbidity when cleaved by the clotting enzyme, but not enough to form a clot (64). Although the solid gel-clot assay is still widely used as an LAL test, it has the disadvantage of being an "early end point" test. Consequently, if it is used, endotoxin cannot be quantified below the level at which a solid clot is formed. The LAL turbidimetric assay, on the other hand, gives a more quantitative measurement of endotoxin over a range of concentrations. This assay is predicated on the fact that any increase in endotoxin concentration causes a proportional increase in turbidity due to the precipitation of coagulable protein (coagulogen) in lysate (hence forming coagulin). Thus, the OD of various dilutions of the substance to be tested are read against a standard curve obtained that has been spiked with known quantities of endotoxin in sterile water.

Kinetic Chromogenic Substrate Assay

The chromogenic assay differs from the gel-clot and turbidimetric reactions in that the coagulogen (clotting protein) is partially (or wholly) replaced by a chromogenic substrate, which is a short synthetic peptide containing the amino acid sequence at the point of interaction with the clotting enzyme. The end of this peptide is bound to a chromophore, para-nitroanilide (pNA).

Japanese workers pioneered the use of chromogenic substrates and lysate (from Limulus and from Tachypleus, the Japanese horseshoe crab) for the detection of endotoxin (65,66). The chromogenic method takes advantage of the specificity of the endotoxin-activated proclotting enzyme, which exhibits specific amidase activity for carboxyterminal glycine-arginine residues. When such sequences are conjugated to a chromogenic substance, pNA is released in proportion to increasing concentrations of endotoxin. Thus, it is possible to measure endotoxin concentration by measuring endotoxin-induced amidase activity as release of chromo-phore. Release of chromogenic substrate is measured by reading absorbance at 405 nm. Testing is conducted with 100 mLe of lysate and an equal amount of sample or diluted sample. The quantitative relationship between the logarithm of the endotoxin concentration and amidase activity can be observed between 5 x 1026 and 5 x 10 ng/mL of endotoxin and, therefore, can be used for the detection of picogram quantities of endotoxin, associated with medical device eluates, immersion rinse solutions, and drug products.

The early chromogenic studies of Lindsay (66) have been extended in recent years by a number of investigators. Suzuki et al. improved the chromogenic assay by using Tos-Ile-Glu-Gly-Arg as a chromophore with the lysate prepared from the amebocytes of Tachypleus (67). Endotoxin-induced amidase activity was optimal at a pH of 8 and at 40°C. Induction of the reaction requires Mg2+. The authors concluded that the system was 50 times more sensitive than the Limulus gelation test. The single step chromogenic method was subsequently developed by Linday et al. Associates of Cape Cod (68) and in this form set the stage for today's simplified use of the kinetic and end point chromogenic assays. The previous chromogenic assays were all end point assays [except for that used by Urbaschek et al. (69) in 1984, which used an experimental kinetic chromogenic test for clinical endotoxemia studies] employing multiple reagent additions that introduce more variability.

The New Recombinant Factor C Reagent

The commercialization of Wang et al. and Tan et al.'s (35,37) efforts to isolate and clone the endotoxin sensitive region of the factor C biosensor has resulted in a remarkable feat: the mass production of a sensitive detector of bacterial endotoxin that is not reliant on a blood source. Interesting detail can be gained from the United States patents on the material/ The method employs a copy of the factor C endotoxin-binding sequence of 333 amino acids of Carcinoscorpius rotundicauda (i.e., Singapore horseshoe crab) DNA expressed in a baculoviral system, which is the first such expression system to preserve the "highly complex mosaic structure" (US patent 6,719,973) required to detect endotoxin. The entry of such a test was able to by pass the level of regulatory scrutiny historically reserved for LAL in that it is not the by-product of a blood system and therefore is not subject to the same type of eThe Seiku Geiku ACC package insert references the use of 50 microliters of sample and standard.

fPatent numbers 5,712,144 and 6,719,973.

FIGURE 6 Typical reactions using the microslide Limulus amebocyte lysate assay. Source: From Refs. 75,76.

overview as LAL. This allowed rapid commercialization of a product (PyroGene™ by Cambrex Biosciences). The removal of such a barrier of entry being seemingly opens the door for eventual cost reduction once the patents expire or if other, similar, recombinant type biosensors are introduced in a competitive manner. The assay, though still relatively new, provides needed insurance against the inevitable, unfortunate decline of the most ancient of mariners (Limulus).

The new rFC assay is incorporated in a process analytical technology employed by the manufacturer Cambrex in their PyrosenseTM automated water testing robot (in-line system) as described in Chapter 16. Seemingly, a barrier to early user-acceptance of rFC appears to be the use of fluorescence instead of the conventional spectroscopy used in the kinetic photometric assays. The limit of detection claim is 0.01 EU/mLg. Though Cambrex has incorporated the test into their software platform a reader with fluorescence capability is a necessity if employing the test. Correspondingly, the validation and qualification activities do not appear as clear cut as those that have become commonplace in LAL testing, given the 1987 Guideline on LAL validation.

"CATS AND DOGS" OF LAL

The methods mentioned here are largely antiquated and would not meet compen-dial requirements for endotoxin testing; nevertheless, they give an appreciation for the current methods and bring with them the knowledge that today's tests are but a fraction of the number of tests that could be (and that have been) developed using the basic LAL mechanism, as discovered by Levin and Bang. Some of the assays also serve as references for reduced-reagent assays the desirability of which may increase with diminished populations of horseshoe crabs, though presumably rFC may negate such a necessity. It is also conceivable that some could be adapted to microfluidics or nanotechnology methods. This information was elaborated on in the first two editions, but has now been condensed into Table 5 given herewith.

gCambrex Biosciences package insert for PyroGene™.

TABLE 5 Largely Superseded Methods

Diazo-Coupling of Chromogenic Reagent

A test not widely used is an extension of the chromogenic assay in that the p-nitroaniline formed by the conventional reaction is further chemically modified by the additional reagents to form the magenta colored, azo dye (62). While only an end point test, it provides one advantage in that the magenta color is read at an absorbance of 540 nm thus avoiding interference with yellow samples such as urine and culture media (71).

The Travenol Optical Density-Lowry Protein-Colorimetric Method

Like turbidimetric, it is based on the observation that increasing concentrations of endotoxin precipitate proportionally increasing amounts of lysate protein. Thus, the amount of protein (coagulin) precipitated can be quantified by performing a simple Lowry protein determination (71). Equal volumes of test sample and lysate (0.1 mL) are mixed in pyrogen-free tubes and incubated at 37°C for one hour and then centrifuged at approximately 1375xgfor 10 mm. Supernatant is then removed by vacuum aspiration. The amount of lysate-specific precipitated protein is determined using the Qyama and Eagle (72) modification of the Lowry protein assay. Using a flow-through spectrophotometer, the OD is recorded at 660 nm. The results can then be related to a standard reference curve.

Nephelometric Method (NM)

NM test has found some utility (i.e., in Japan) as a means of pyrogen detection in large-volume parenterals (LVP) and testing water for production or rinses of depyrogenated components (73). The method employs a Hyland laser nephelometer. Instead of being read as OD, the nephelometer records the light scattered relative to its position at a 90° angle from the light source (Tyndall scattering of turbid liquids) (74). Sodium dodecyl sulfate (SDS) terminates the enzymatic reaction and ensures the uniformity of precipitated lysate-specific protein. Critical micellar concentration (CMC) is then added as a suspension stabilizer. The test can be completed within an hour using only 50 mL of lysate, which can readily detect picogram amounts of endotoxin.

Slide Test

Frauch (75) first suggested a simple LAL slide test. Using a calibrated capillary pipette, 10 mL of lysate was mixed with an equal volume of sample. This preparation is incubated in a moist chamber at 37°C for 30 minutes. A positive control, a negative control, and test samples were prepared on slides with black backgrounds. The controls and samples are easily differentiated on the basis of viscosity and turbidity. Another Japanese worker, Okuguchi (76), reported an improved LAL microslide method employing 10 mL of lysate. Samples are incubated in the presence of lysate on a tissue culture chamber slide for 30 minutes at 37°C. Test samples are then stained with a drop of bromophenol blue and end points determined using an inverted-phase contrast microscope. Samples forming a ring filled with cell debris are negative for endotoxin, but positive samples exhibit a cloud-like formation throughout the mixture.

Micro Assay

Gardi and Arpagaus (77) described an LAL microtechnique using 1 mL of reagent and sample. Samples are incubated in 5 mL capillary tubes, which are dipped into a dye. When a firm gel is formed, the dye cannot enter the tube and the test result is recorded as positive. The single greatest virtue of the microslide method is cost savings; the price of lysate has historically been considerably higher elsewhere than in the United States.

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