Abstract
The only definitive management of snake envenoming is the use of snake antivenom. Endotoxin contamination is a serious threat to the safe use of parenteral drugs. A greater understanding of the nature of limulus amebocyte lysate (LAL) test interference and use of permissible dilutions has minimized enhancement problems. Common interference issues include suboptimal pH, enzyme or protein modification, and nonspecific LAL activation. This study aimed at determining the interference factors associated with validating the antivenom sera preparations to avoid false-positive results when testing snake antivenom serum samples by the LAL method. Phase I (preliminary screening/interference assay) was performed to determine a compatible test dilution, which was then used in Phase II (inhibition-enhancement/validation study). The best approach to resolve interference issues was dilution by 1:80 (maximum valid dilution) plus a specific treatment as heat-activation at 70°C–80°C for 10 min with rehydration of LAL reagent with endotoxin-specific buffer solution.
LAY ABSTRACT: Snake antivenom sera are produced by immunizing horses with repeated nonlethal doses of snake venom. Bacterial endotoxins constitute one of the major problems in the formulation of pharmaceutical products. One such method for detecting endotoxin levels is the bacterial endotoxin test (BET). However, some substances show strong interfering action with the BET that cannot be avoided by simply diluting the sample solution. In this work, the test for interfering factors was performed as two identical series of product dilutions—one spiked with 2λ and one left unspiked. The result of the interference test revealed the noninterfering dilution (NID) of the product, which was used for the actual validation. Our results showed that after treating the samples using different procedures, such as heat activation at 70–80°C for 10 min followed by centrifugation at 2000 rpm for 10 min and dilution of samples in BD100 (biodispersing agent), inhibition and enhancement up to 1:100 maximum valid dilution (MVD) were observed. Finally, to resolve this inhibition/enhancement problem, the activated sample was heated at 70–80°C for 10 min with rehydration of the Endosafe LAL reagent in an endotoxin-specific buffer solution (BG120) to block β-d-glucans and limulus amebocyte lysate (LAL) reactive material (LAL-RM).
- Polyvalent horse snake antivenom
- Endotoxin
- LAL
- Validation study
- Maximum valid dilution
- Interference factors
Introduction
Snakebite incidents with their associated complications and fatalities are a major public health concern worldwide, particularly in certain developing regions, such as Southeast Asia, sub-Saharan Africa, and Latin America. Snakebite is an ongoing problem that usually occurs in children and people working in fields or on farms (1⇓–3). Although the number of deaths related to this problem might reach tens of thousands each year, there has been little progress associated with dealing with such a devastating hazardous issue (4). The antivenom serum is the specifically used in the treatment of snake envenoming. Antivenoms are produced by the fractionation of plasma usually obtained from large domestic animals hyperimmunized with repeated nonlethal doses of snake venoms. After the animals are immunized, their plasma is subjected to fractionation and extraction of immunoglobulins. The range of extracted products includes intact immunoglobulin G, separated by ammonium sulfate or caprylic acid, F(ab′)2 prepared by pepsin digestion and ammonium sulfate or caprylic acid fractionation, or F(ab) prepared by papain digestion and ammonium sulfate fractionation (5, 6).
Bacterial endotoxins (ETs) are lipopolysaccharides (LPSs) present in the outer cell surface of Gram-negative bacteria and constitute one of the major problems in the formulation of parenteral pharmaceutical products (7). Endotoxins are large-molecular-weight lipopolysaccharide complexes with molecular weights of ∼50 to ∼100 kDa. They are usually associated with, and shed from, the outer membranes of Gram-negative bacteria; endotoxin effects include the induction of fever, headache, and severe hypotensive shock (8). Consequently, one of the first quality check points before the release of any parenteral drug in any market is to check for the presence of endotoxins.
In the late 1960s, Levin and Bang (9) developed an endotoxin test that involved mixing the blood-clotting factors obtained from amebocytes with a drug sample in a test tube. If sufficient endotoxin was present the liquid in the tube would clot. This was the first “LAL” test for endotoxin. LAL stands for limulus amebocyte lysate, which contains factors inside the amebocytes of the Atlantic horseshoe crab, Limulus polyphemus.
In 1987, the Food and Drug Administration (FDA) (10) approved the use of the LAL test as a replacement for the rabbit pyrogen test to detect endotoxins in human- and animal-injectable pharmaceuticals and biologicals as well as implantable medical devices. It is worth mentioning that the test using lysate from the Atlantic horseshoe crab amebocytes is 3–300 times more sensitive than the rabbit pyrogen test (11).
One of the most time-consuming aspects of endotoxin testing using LAL is pretreating samples to overcome assay inhibition and enhancement. Certain requirements are needed for the reaction of the LAL with the endotoxin, including neutral pH, optimum Na+ and divalent cations levels, and a uniform temperature of 37°C. In most cases, dilution with LAL reagent water (LRW) is usually needed before testing to avoid interference. Any pretreatment of a specimen deemed necessary to conveniently overcome interference requires validation using no less than three batches of each product or test specimen (12).
Two major problems that are associated with the LAL test include inhibition and enhancement. Positive product control (PPC) test is a common test for assessing inhibition. Enhancement can be caused by substances such as (1-3)-β-d-glucan and trypsin that give a positive reaction and is sometimes inappropriately referred to as “false positive,” a term that should not be used, as it is always the endotoxin that gives the positive result (13).
There are various reasons for interference, including suboptimal pH, endotoxin modification, container effects, unbalanced cation levels, protein or enzyme modifications, and finally, non-specific LAL activation.
A neutral pH is essential for LAL reaction and is usually achieved by adding 0.1 N HCl or 0.1 N NaOH for basic and acidic products, respectively. Endotoxin modification occurs when the endotoxin forms micelles because of the hydrophilic and hydrophobic interaction between LPS and water, which results in the endotoxin escaping the LAL test. Such a problem is alleviated by vortexing the samples to achieve proper mixing or by adding a dispersing agent. To avoid container effects, the use of a high-quality borosilicate glass tube is recommended because of its inert nature and to avoid any adsorption of the endotoxin. The divalent cations Ca2+ and Mg2+ can be added externally to achieve optimum cation levels that are needed for LAL reactivity and endotoxin detection. Oxidants, proteolytic agents, or inactivators will lead to protein or enzyme modification with subsequent inhibition. Enhancement, which is still a rare reaction, can occur if some molecules other than the endotoxin react with the LAL reagent to generate a gel product (14).
Dilution may help, particularly when the maximum valid dilution (MVD) is large. Heat treatment at 70°C–80°C for 5 min is commonly used to denature the protein in the sample and allow the heat-tolerant endotoxin to be detected. Roth et al. (15) found that a fourfold dilution of plasma with 0.15 M NaCl followed by a 30-min heat treatment at 60°C was the most effective procedure. A wide range of other treatments of blood, plasma, and serum have been described, including use of acids, bases, organic solvents, and surfactants, either alone or in combination (15).
In the biotech and research fields, interference is best avoided by dilution of the LAL reagent with water, a procedure that is usually performed while testing most biotherapeutic products, as serum, plasma, and protein samples are subject to inhibition because of the presence of serine protease inhibitors (16, 17). For routine testing of products, the product must be prepared in the manner in which it was treated to pass the inhibition/enhancement test (18). Otherwise, a negative gel clot assay may be mistaken as indicating a lack of endotoxin in the product when in reality the negative is a result of inhibition (19). The aim of this study is to determine the interfering factors that lead to the diverse results in snake antivenom immunoglobulins and severe complications in patients upon administration of these antivenoms.
Materials and Methods
Multitest LAL Reagent 5.2 mL/vial, sensitivity 0.03 endotoxin units/mL (EU/mL), LAL reagent water (LRW), control standard endotoxin (CSE) 7500 EU/vial (to bracket the labeled sensitivity of the lysate), 0.1 M Tris HCl (BT102), 0.25 M Tris Base (BT101), Endosafe dispersing agent (BD100, 0.05% (v/v) phosphate ester surfactant), and endotoxin-specific buffer solution (BG120, carboxymethylated curdlan) were purchased from Charles River (Charleston, USA). Depyrogenation of glassware was carried out by heating in a hot-air oven for 30 min at 250°C. Micropipette tips were free from pyrogen. All chemicals were purchased from Sigma Aldrich.
Preparation of Equine Polyvalent Antivenoms
Polyvalent snake antivenom plasma was obtained by mixing monospecific hyperimmune plasma antivenom prepared separately against three snake venoms (Cerastes cerastes, Naja haje [cobra], Naja nigricollis) in horses belonging to Helwan farm, The Egyptian Company for Production of Vaccines, Sera & Drugs (EGYVAC). These hyperimmune sera were fractionated by ammonium sulfate precipitation with pepsin digestion to obtain F(ab′)2 fragments. The refined antivenoms contained interfering factors as contaminating heterologous serum proteins, Fc, serine proteases inhibitors, and other fragments and aggregates.
Preparation of Sample Solution for LAL
Using aseptic techniques, a sterile, endotoxin-free final snake antivenom immunoglobulin product was diluted to the required concentrations based on the MVD, which is the maximum allowable dilution of the specimen at which the endotoxin limit can be determined and calculated: MVD = (endotoxin limit × concentration of sample solution)/(λ), where λ is the labeled sensitivity of LAL reagent. The endotoxin limit (EL) of the test sample is specified in the individual monograph in terms of volume or units of active drug (in EU/mg) and listed in Appendix E in (10) or defined on the basis of dose, which is equal to K/M (K is the threshold pyrogenic dose of endotoxin per kilogram of body mass; M is the maximum recommended bolus dose of product per kilogram of body mass). For this study K was 5 EU/kg, M was 60/70 mL/kg, EL = K/M = 5.83 EU/mL, MVD = EL/λ = 5.83 EU/mL/0.03,125 EU/mL = 186. When possible contamination from water for injection is considered, EL was calculated as 3.58 EU/mL, and MVD was 114. The following test dilutions were prepared by dilution with LRW or BD100 (endotoxin-dispersing agent) as 1:10, 1:20, 1:40, 1:80, and 1:100.
Preparation and Reconstitution of LAL Reagent
Lyophilized lysate was reconstituted by adding 5.2 mL LRW or endotoxin-specific buffer solution directly into the vial just before use. The LAL reagent was mixed gently for at least 30 s.
Preparation of Standard Stock Endotoxin Solution
The control standard endotoxin (CSE) having a defined potency of 7500 EU/vial as specified on the certificate of analysis (CoA) was reconstituted with 7.5 mL of LRW to obtain 1000 EU/mL.
Preparation of Standard Endotoxin Solutions
After mixing the CSE, a series of twofold dilutions of the endotoxin standard in LRW were prepared to give final concentrations of 2λ, λ, 0.5λ, and 0.25λ.
Test for Confirmation of Labeled LAL Reagent Sensitivity
The test was performed on each lot of LAL-CSE received. From each concentration of the standard solutions, 100 µL was added separately into depyrogenated 10 ×75 mm LAL-grade tubes and labeled accordingly. As a negative control 100 µL LRW was added separately into depyrogenated LAL-grade tubes. Each dilution, as well as the negative control, was performed in quadruplicate. Then 100 µL of reconstituted lysate was added into each tube and mixed gently. All tubes were incubated in a noncirculating water bath at 37°C ± 1°C for 60 ± 2 min.
After incubation, each tube was carefully removed and gently inverted at 180°C. The end point was the last positive result in the series of decreasing concentrations of endotoxin. The geometric mean (GM) end-point concentration of the solutions was determined using the equation GM = antilog (Σe/f), where Σe is the sum of the log end-point concentrations of the dilution series used and is the number of replicate test tubes. The geometric mean end-point concentration was the measured sensitivity of the lysate solution (EU/mL), which must be in the range of 0.5λ–2λ (20).
Validation of the Gel Clot LAL Method for the Finished Product
A preliminary screening/interference assay (Phase I) was performed on one batch of product to determine a compatible test dilution/concentration, which was then used in the inhibition enhancement/validation study (Phase II), in which three batches of product were tested at this compatible test dilution/concentration. The GM of the endotoxin standard series in both the optimal (compatible) sample dilution and LRW can be achieved with the same efficiency (14, 21).
Phase I (Preliminary Screening/Interference Assay)
A standard curve of CSE in LRW was prepared using the 2λ and 20λ standards. A range of sample dilutions were tested for interference including testing the undiluted product. All tubes were labeled for 2λ assay-positive water control (PWC) and negative LRW water control (NWC); each sample dilution included undiluted negative product control (NPC), and each sample dilution included undiluted positive product control (PPC). Two replicates were performed.
The test specimen (100 µL) was inoculated into depyrogenated LAL-grade tubes; a PPC was prepared by adding 10 µL of 20λ standard into two tubes for each concentration-spiking method. To perform PWC and NWC, 100 µL of 2λ assay standard and LRW, respectively, were added into two tubes each. Then 100 µL of reconstituted LAL reagent was added to each labeled tube.
The pH of the product/LAL mixture at the compatible dilution/concentration was adjusted to between 6.5 and 8.0, if necessary, using 0.1 M Tris HCl solution or 0.25 M Tris Base solution not exceeding 10% of the sample volume. All tubes were placed in a noncirculating water bath at 37°C ± 1°C for 60 ± 2 min. After the incubation period, each tube was carefully removed, one at a time, and inverted slowly toward 180°.
Phase II (Inhibition Enhancement/Validation Study)
A standard curve of CSE or reference standard endotoxin in LRW was prepared for each batch to be tested (three batches were required for a full validation); a 6 mL sample at twice the dilution (noninterfering dilution, NID) that was previously determined in the interference screen was prepared.
A standard curve for each batch of product was determined as follows:
PPC and PWC were created by inoculating 100 µL of each test specimen and each concentration of standard in water, respectively, into each of four depyrogenated LAL grade tubes; 100 µL of compatible dilution of product and LRW was transferred into each of the four reaction tubes to prepare negative product control (NPC) and negative water control (NWC), respectively.
Reconstituted LAL reagent 100 µL was added to each labeled tube. The pH of the product/LAL mixture at the compatible dilution/concentration was adjusted to between 6.5 and 8.0. All tubes were placed in a noncirculating water bath at 37°C ± 1°C for 60 ± 2 min. After the incubation period, each tube was carefully removed and inverted slowly toward 180° The results were valid if all tubes containing negative controls were without gels and the end point of the standard curves in water and product was not greater than a twofold dilution to either side of the lysate sensitivity and the end point of the standard curve in the product was not greater than a twofold difference to the end point of the standard in water. If the above criteria were met, the product had been validated for testing in the LAL gel clot assay.
Results
Test for Confirmation of Labeled LAL Reagent Sensitivity (0.03,125 EU/mL)
The GM end-point concentration of the solutions was determined for two lots received. For the second lot number (H0K356), the GM end-point concentration of the solutions was determined using the equation GM = antilog (Σe/f) = 0.0301 EU/mL.
Validation of Gel Clot LAL Method for Finished Product
Phase I: Preliminary Screening/Interference Study:
In this study two series of samples at various dilutions were used. One was left unspiked and the other was spiked with the equivalent of 2λ. Results of this phase should show the NID) of the product, which is the first set of PPC that shows gel formation. Test samples at dilutions of 1:10, 1:20, 1:40, 1:80, and 1:100 in LAL reagent grade water were heated at 70°C–80°C for 10 min to coagulate the proteins followed by centrifugation at 2000 rpm for 10 min to remove debris. Table I showed enhancement up to 1:100 (MVD).
LAL Assay Results of Snake Antivenom After Heat Denaturation
Test samples were also prepared using BD100 dispersing agent instead of LRW to dissociate and disperse the endotoxin molecules from complex biological products; samples were vortexed vigorously for at least 5 min between each dilution to prepare 1:10, 1:20, 1:40, 1:80, and 1:100 followed by centrifugation at 2000 rpm for 10 min to remove debris. Assay results are presented in Table II.
LAL Assay Results of Snake Antivenom After Dilution with BD100 Dispersing Agent
Tables II and III showed enhancement up to 1:100 (MVD). To sort out this problem, the sample was heat-activated at 70°C–80°C for 10 min to coagulate the proteins. In addition, the Endosafe LAL reagent was rehydrated with endotoxin-specific buffer solution (BG120) to block β-d-glucans and LAL-reactive material (LAL-RM), and the results are presented in Table IV.
LAL Assay Results of Nonheat-Activated Snake Antivenom After Rehydration of LAL Reagent with Endotoxin-Specific Buffer Solution
LAL Assay Results of Heat-Activated Snake Antivenom and Rehydration of LAL Reagent with Endotoxin-Specific Buffer Solution
Table IV shows enhancement up to 1:20 (MVD). Therefore, the NID was found to be 1:40. It was advisable to validate the product at not less than 1:80 dilution to take care of any batch-to-batch variation during regular production. So, 1:80 dilution was chosen for product validation. Once a NID was identified, Phase II (inhibition enhancement/validation study) was performed.
Phase II: (Inhibition Enhancement/Validation Study):
Endotoxin dilutions were prepared as two identical series bracketing λ, one prepared in LRW and the other prepared in the product diluted to the proposed test dilution after appropriate treatment. The dilution selected for validation was 1:80 (hot spike method). Three batches of product were tested at this compatible test dilution/concentration.
The validation test results presented in Tables V and VI were considered valid because the gel end-point values for each series of CSE dilutions in water and CSE dilutions in product are within a twofold dilution of each other and a twofold dilution of the claimed LAL sensitivity, all tubes containing negative controls were without gels, and the pH of the product–lysate mixture was within the acceptable range of 6.0–8.0. Therefore, the product was validated for testing in the LAL gel clot assay and validation was conducted at this dilution on two other batches of product, which showed nearly the same results.
Labeled LAL Reagent Sensitivity for Sample Matrix (Endotoxin/Product)
Labeled LAL Reagent Sensitivity for Water Matrix (Endotoxin/LAL Reagent Water)
Discussion
A major sociomedical dilemma that is still affecting certain communities in developing countries in Africa and Asia is snakebite, which is still treated by administration of products previously prepared in horses or sheep. Such preparations aim at neutralizing the toxins. Till now, these serum-based preparations are the sole treatment options for such bites. They are usually the F(ab) of IgG that has been purified from a horse or sheep immunized with one or more venoms from the different species of snakes known to be present at a particular geographic location (22). Our polyvalent antivenom was produced by immunizing individual horses with venom of a single species (C. cerastes, N. haje [cobra], and N. nigricollis) and mixing the various hyperimmune plasma for fractionation and formulation.
Polyspecific products offer a great advantage over monospecific ones, as they eliminate the need for prior identification of the particular snake species before initiating therapy and therefore simplify treatment (23).
The outer lipopolysaccharide membrane present in Gram-negative bacteria triggers a cascade of inflammatory reactions that result in fever. Endotoxins are usually small-molecular-weight macromolecules that are ∼20–30 KD and are negatively charged. They vary in size because of their bacterial origin or the presence of divalent cations or biological detergents. Endotoxins are pyrogens that can contaminate parenteral products (14). It is necessary to determine and control the levels of endotoxin contamination in injections as a quality-control measure (24).
An alternative method to the rabbit pyrogen test, which measures the inflammatory response in an intact animal (rabbit) to the pyrogen, is the limulus amebocyte lysate or LAL test, which detects pyrogenic material in sterile preparations. However, care should be taken when carrying out such a test to avoid interference that may obscure the actual results. Interference can take the form of inhibition or enhancement (false positive) (25).
Inhibition is caused by any material known to denature protein or to inhibit enzyme action; this can be overcome by dilution or pH adjustment. Of course, dilution reduces the concentration of the endotoxin and places greater demand on the sensitivity of the LAL reagent to detect diluted amounts of endotoxin.
Tests for activation or inhibition necessitate the use of positive controls. In these tests, samples are “spiked” with an endotoxin of known concentration or level, usually at the same dilution as the standard used to determine sensitivity. There should be no difference between the two end points of the product samples and the standard series (17).
To adequately and properly test polyvalent snake venom antiserum samples by the LAL method, the samples must not inhibit or enhance the gelation response, otherwise it will interfere with the LAL assay. Therefore, the first step in creating a LAL test method for a pharmaceutical entity is the identification of a compatible sample concentration for routine testing (10). Thus, the establishment and validation of the specifications for the quality control of the medicinal products tested is recommended.
In this study, we aimed to optimize different conditions to perform the LAL test using the gel clot method. The interference in the LAL test could be either inhibitory or false-positive interference effect. The inhibitory interference might result from high concentration of the sample itself. Therefore, in this study, confirmation of lysate sensitivity was carried out on two lots of LAL-CSE. The test results were shown to be valid as the lowest concentration of the standard solution (0.25λ) showed a negative result in all replicate tests and the GM end-point concentration was the measured sensitivity of the lysate solution (EU/mL) for the two lots, so the labeled sensitivity of 0.03,125 EU/mL was confirmed with this lysate.
Two phases of sample dilution tests were carried out (Phase I and Phase II). Phase I was performed on a 10-fold serial dilution series of the product. Phase II was performed through a twofold serial dilution of the sample allowing the accurate determination of the dilution at which interference was eliminated.
To avoid interfering test conditions, the USP allows drug product dilutions based on the established endotoxin limits (18). The MVD was calculated using the equation MVD = (EL × concentration of sample)/(λ); therefore MVD = 1:114. The MVD factor so obtained is the limit dilution factor for the preparation for the test to be valid.
As shown in Tables I⇑–III, enhancement up to 1:100 (MVD) was detected. The interference in the LAL test could be false-positive interference (enhancement) that results from the coagulation of the protein content in the sample, LAL-RM in plasma and not from endotoxin, and contamination as the sample is endotoxin free (treatment with affinity chromatography).
Finally, to sort out this enhancement problem, the activated sample was heated at 70°C –80°C for 10 min with rehydration of the Endosafe LAL reagent in endotoxin-specific buffer solution (BG120) to block β-d-glucans and LAL-RM. These findings nearly agree with Pennamareddy et al. (14), who stated that heat denaturation was effective in solving interference problems in biotherapeutic drugs.
Poole (26) stated that erythropoietin and liver albumin injections give false-positive results because of the coagulation of proteins at 37°C while performing the LAL test. This false-positive interference in the LAL test could be eliminated by heating at 90°C for 15 min to coagulate the protein before performing the LAL test, which agrees with our results.
False-positive LAL test results have been associated with intravenous immunoglobulin preparations. The amount of LAL-RMs increases as the amount of administered immunoglobins increases. A substance called (1-3) β-d-glucan-sensitive factor has been found to be associated with the LAL reagent and can activate it to produce a false-positive result for the presence of endotoxin. These false reactions caused by non-endotoxin products have been previously addressed by Ohno et al. and Pearson and Weary (27, 28).
Another accepted procedure by Donovan (19) for sera is the exposure of the sample to boiling water for 2 or 3 min before further dilution to at least 10-fold. Also, Cooper (17) mentioned that a biological product that contains a serine protease is LAL-reactive under certain conditions. Heat treatment to denature the enzyme before the LAL assay will readily eliminate this type of false-positive LAL activity. In particular, the specificity of lysate reagents to detect endotoxin was improved by the removal or suppression of factor G in lysate reagents, which eliminated the reactivity to β-d-glucan and other nonpyrogenic substances (29).
Phase I results revealed that the NID is 1:80, which was then chosen for product validation (Phase II). The validation test results obtained were considered valid because the negative controls did not gel, the positive controls formed a firm gel, and pH measurement was 7 as shown in Tables V and VI. Therefore, there is no possibility of false-positive results, and the same methodology could be applied for other similar products.
Conclusions
We present this study as an attempt to help all personnel working in the pharmaceutical industry to avoid the common problems encountered while performing the LAL test on parenteral products. Heat activation at 70°C–80°C for 10 min and rehydration of the LAL reagent with endotoxin-specific buffer solution is the best method to manage the enhancement problem. In addition, the use of a more sensitive LAL reagent or test method can help to increase the MVD.
Conflict of Interest Statement
The authors declare that they have no competing interests. All authors read and approved the final manuscript.
Acknowledgment
The authors would like to thank Dr. Nabil El Biblawy, Chairman and C.E.O of The Egyptian Company for Production of Vaccines, Sera & Drugs (EGYVAC) for his tremendous support during this work.
- © PDA, Inc. 2019
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