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org on March 16, 2019

A Validation study of the Limulus Amebocyte Lysate test as an

end-product endotoxin test for Polyvalent Horse Snake
Antivenom
Norhan Saif Sheraba, Mohamed Reda Diab, Aymen Samir Yassin, et al.

PDA Journal of Pharmaceutical Science and Technology 2019,

Access the most recent version at doi:10.5731/pdajpst.2018.009522
 Downloaded from journal.pda.org on March 16, 2019

A Validation study of the Limulus Amebocyte Lysate test as an end-product

endotoxin test for Polyvalent Horse Snake Antivenom

Norhan S. Sheraba1,2, Mohamed R. Diab2, Aymen S. Yassin3, Magdy A. Amin3 and Hamdallah H.
Zedan3
1
Department of Pharmaceutics, College of Pharmacy, King Khalid University, Asir, Abha, Kingdom
of Saudi Arabia
2
VACSERA, the Holding Company for Biological Products and Vaccines,

Giza, Egypt
3
Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

*Corresponding author: Norhan S. Sheraba, PhD, Department of Pharmaceutics, College of

Pharmacy, King Khalid University, Asir, Abha, P.O.Box : 960, Postal Code : 61421, Kingdom of
Saudi Arabia; Phone number: 011+ 966+ 172418000; Mobile number: +966 583637720: Email:
[email protected], [email protected]

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ABSTRACT: Snake antivenoms are the only definitive management of snake envenoming’s.
Endotoxin contamination is a serious threat to the safety of parenteral drugs. A greater understanding
of the nature of LAL-Test interferences and the use permissible dilution has minimized enhancement
problems. Common interference mechanisms include suboptimal pH, enzyme or protein modification,
and non-specific LAL activation. The study aimed at sorting out the interfering factors before
validating the antitoxic sera preparations to avoid false positive results when testing snake venom
antiserum samples by (LAL) method. Phase I (Preliminary Screening /Interference Assay): was
performed to determine a compatible test dilution, which is then used in Phase II (Inhibition-
Enhancement / Validation Study). Dilution is the best approach to resolve interferences by 1:80
(MVD) plus a specific treatment as heat-activation of at 70-80°C for 10 minutes with rehydration of
LAL reagent with Endotoxin Specific Buffer Solution to sort out the enhancement problem.

KEYWORDS: Polyvalent Horse Snake Antivenom; Endotoxin; LAL; Validation Study; Maximum
Valid Dilution; Interference factors.

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 on BET which 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 interference test will reveal the non-interfering dilution (NID) of the product, which was used
for the actual validation. Our results showed that after analyzing the sample using different procedures
such as heat-activation at 70-80°C for 10 minutes followed by centrifugation at 2000 rpm for 10
minutes; dilution of sample in BD100 (Bio-dispersing agent), both inhibition and enhancement up to
1:100 Maximum Valid Dilution (MVD) were observed. Finally, in order to resolve this
inhibition/enhancement problem, the activated sample was heated at 70-80°C for 10 minutes with
rehydration of the Endosafe LAL reagent in Endotoxin-Specific Buffer Solution (BG-120) to block β-
D-Glucans and LAL-Reactive Material (LAL-RM).

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Introduction:

Snake envenoming and the associated morbidity and mortality is one of the important public
health problems in rural tropical areas like Southeast Asia, sub-Saharan Africa, Latin America. Snake
bite is a neglected, environmental and occupational disease, which primarily affects agricultural
workers and children (1-3). It has been estimated that around 20,000 to 94,000 people die of snake bite
each year (4). Antivenom serum is the only specific treatment for envenoming by snakebites. They are
produced by the fractionation of plasma usually obtained from large domestic animals hyper-
immunized with repeated nonlethal doses of snake venoms. After the immunization, the plasma of
animals is subjected to fractionation and extraction of immunoglobulin substances. These can be intact
IgG, isolated using either ammonium sulphate or caprylic acid or F(ab’)2 fragments, with pepsin
digestion and ammonium sulphate or caprylic acid fractionation; and Fab fragments, prepared from
papain digestion and ammonium sulphate fractionation (5, 6). Upon preparation when highly stringent
measures against microbial contamination are not taken, the horse hyperimmune snake antivenom sera
might be contaminated by endotoxins from accidental microbial contamination (7).

Bacterial endotoxins (ET) are lipopolysaccharides (LPS) present at the outer cell surface of gram-
negative bacteria and constitute one of the major problems in the formulation of parenteral
pharmaceutical products. Endotoxins are large molecular weight complexes (~106 Da) associated
with, and shed from, the outer membranes of Gram-negative bacteria; endotoxin interactions include
the induction of fever, headache and severe hypotensive shock (8). Consequently, it is mandatory to
check the presence of endotoxin level in parentral drugs before releasing the product into market.

In the late 1960s, Frederick Bang and Jack Levin (9) developed an endotoxin test that involved
mixing the blood-clotting factors that are in the amebocyte 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 and contains the factors inside the amebocytes
of the horseshoe crab, Limulus polyphemus.

In 1987, Food and Drug Administration (FDA) (10) approved the use of LAL test as a
replacement for the Rabbit Pyrogen to detect endotoxin in human and animal injectable
pharmaceuticals and biologicals, and implantable medical devices. It is worth mentioning that lysate
from washed Amoebocyte of horseshoe crab is 3-300 times more sensitive than rabbit pyrogen test
(11).

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One of the most time-consuming aspects of endotoxin testing using LAL is pretreating samples to
overcome assay inhibition and enhancement. The LAL reaction with endotoxin requires pH neutrality
and optimum levels of Na+ and divalent cations. A uniform temperature of 37° C optimizes the rate of
reaction. Most therapeutic drug products require dilution with LAL reagent water (LRW) 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).

The positive product controls (PPCs) is routine gel-clot tests only control for inhibition, not
enhancement. Two notes about enhancement: firstly, the phenomenon is sometimes referred to as a
"false positive". It is recommended that the term be restricted to substances other than endotoxin that
give a positive LAL test, such as (1→3)-β-D-glucan and trypsin. Secondly, a positive, valid endotoxin
test should be treated as contamination. "Enhancement" should never be used as a euphemism for
contamination (13).

Interference may occur due to following reasons: 1) suboptimal pH: The optimal pH for LAL
reaction is in between about 6.8 to 7.4. Hence it is very necessary to bring pH of reaction to neutral
range. This can be done using 0.1N HCl or 0.1N NaOH for basic and acidic product respectively. 2)
Endotoxin modification: Purified endotoxin has tendency to form micelle formation which is due to
hydrophilic and hydrophobic interactions between LPS and water. This aggregated endotoxin escapes
LAL test. Hence to avoid such problem vortex mixing for samples is performed or addition of
dispersing agent. 3) Container effects: Adsorption of endotoxin on tube wall causes poor recovery of
endotoxin in LAL test. So it is suggested to use high quality borosilicate glass tubes because of its
inert nature hence adsorption of endotoxin is least. 4) Unbalanced cations levels: Divalent cations play
important role in LAL reactivity and dispersion of endotoxins. LAL test requires optimum
concentration of Ca++and Mg++ ions. If divalent cations are insufficient then Ca++and Mg++ ions are
added externally. 5) Protein or enzyme modification: due to oxidants, proteolytic agents or specific
inactivators will cause inhibition. Dilution may help, particularly when the MVD is large. Heat
treatment at 70-80 oC for 5 minutes is commonly used to denature the protein in the sample and allow
the heat tolerant endotoxin to be detected (14). Roth et al. (1990) found that fourfold dilution of
plasma with 0.15 M NaCl followed by a 30-minute heat treatment at 60oC to be the most effective. 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). 6) Non-specific
LAL activation: Some molecules other than endotoxin are known to react with LAL reagent and give
gel formation. This is enhancement reaction and is very rare (16).

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Most Biotherapeutic products require dilution with LAL reagent water (LRW) before testing to
avoid interference. Testing of serum, plasma, protein sample is subjected to inhibition from serine
proteases inhibitors and the interference creates problems in both Biotech and research fields (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 the study is to sort out the interfering factors which 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 5.2mL/vial sensitivity 0.03 Endotoxin Unit/mL (EU/mL); LAL Reagent
Water (LRW), Control Standard Endotoxin (CSE) (7500EU/vial) to bracket the labelled sensitivity of
the lysate; 0.1 M Tris HCl (BT102); 0.25 M Tris Base (BT101); BD100 Endosafe Dispersing Agent
[0.05% (v/v) phosphate ester surfactant]; Endotoxin-Specific Buffer Solution (BG120)
[Carboxymethylated Curdlan] were purchased from Charles River Endosafe, USA). Depyrogenation
of glassware was carried out by heating in 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 were obtained by mixing monospecific hyperimmune plasma
antivenom prepared separately in horses belonging to Helwan farm, Egyptian Company for Sera,
Vaccines and Drugs (EGYVAC), against three snake venoms (Cerastes cerastes, Naja haje (cobra),
Naja nigricollis). These hyperimmune serum were fractionated by ammonium sulphate precipitation
with pepsin digestion to obtain (Fab')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 technique, a sterile, endotoxin-free final snake antivenom immunoglobulins product
was diluted to the required concentrations based on the formulae MVD, which is the maximum
allowable dilution of the specimen at which the endotoxin limit can be determined and calculated;
MVD = Endotoxin limit x Concentration of sample solution)/ (λ). The endotoxin limit (E.L) of the test
sample is specified in the individual monograph in terms of volume or units of active drug (in EU/mg)
listed in Appendix E, contained in (10) or defined on the basis of dose, which is equal to: K/M (K=
threshold pyrogenic dose of endotoxin per kilogram of body mass, M= maximum recommended bolus

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dose of product per kilogram of body mass),  is the labeled sensitivity of LAL reagent. K = 5 EU/kg,
M = 60/70 mL/kg, E.L = K/M = 5.83 EU/mL, MVD = E.L/lambda = 5.83 EU/mL/0.03125 EU/mL =
186. When possible contamination from WFI is considered, E.L was calculated as 3.58 EU/mL, and
MVD was 114.The following test dilutions were prepared by dilution in LAL reagent water 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 LAL reagent water or BG120 (Endotoxin-
Specific buffer) just before use directly into the vial. The LAL reagent was mixed gently for at least 30
seconds.

Preparation of Standard Stock Endotoxin Solution:

The control standard endotoxin (CSE) having a defined potency of 7500EU/vial as specified on
the certificate of analysis (CoA) was reconstituted with 7.5 mL of LAL reagent grade water to obtain
1000 EU/mL.
Preparation of Standard Endotoxin Solutions:
After mixing the CSE, a series of two- fold dilutions of the endotoxin standard in LAL reagent
water were prepared to give final concentration of 2, 0.5 and 0.25.

Test for Confirmation of Labeled LAL Reagent Sensitivity:

The test was performed on each lot of LAL-CSE received. 100 µL from each concentration of the
standard solutions was added separately into depyrogenated 10 X 75 mm LAL grade tubes and labeled
accordingly. For negative control 100 µL LRW water separately was added into depyrogenated LAL
grade tubes. Each dilution, as well as a negative control was performed in quadruplicate. 100 µL of
reconstituted Lysate was added into each tube and mixed gently. All tubes were incubated in non-
circulating water bath at 37 ± 1°C for 60 ± 2 minutes.

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 concentration of Endotoxin. The geometric mean
(GM) endpoint concentration of the solutions was determined using the equation GM = antilog (Σe/f),
where: Σe is the sum of the log endpoint concentrations of the dilution series used, and f is the number
of replicate test tubes. The geometric mean end-point concentration was the measured sensitivity of
Lysate solution (EU/mL) which must be in the range of (0.5-2) (20).

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Validation of Gel Clot LAL Method for Finished Product:

Phase I (Preliminary Screening /Interference Assay): was performed on one batch of product to
determine a compatible test dilution/concentration, which is then used in Phase II (Inhibition
Enhancement / Validation Study) where three batches of product were tested at this compatible test
dilution/concentration. Geometric mean of endotoxin standard series in both the optimal (compatible)
sample dilution and LRW can be achieved with the same efficiency (21, 22).

Phase I (Preliminary Screening /Interference Assay):

A standard curve of CSE or RSE in LAL reagent water was prepared, where the 2 and 20
standards were used. A range of dilutions of sample were tested for the interference screen including
testing the product undiluted. All tubes were labelled for: 2 assay positive control (PWC), negative
LRW water control (NWC), each sample dilution included undiluted negative product control (NPC)
and each sample dilution included undiluted positive product control (PPC) and performed in two
replicates.

100 µL of test specimen was inoculated into depyrogenated LAL grade tubes; a positive product
control was prepared by adding 10µL of 20 standard into 2 tubes for each concentration–spiking
method. 100µL of 2 assay standard and also of LRW were added into each of 2 tubes to perform
positive (PWC) and negative water control (NWC) respectively. 100µL of reconstituted LAL reagent
was added to each labeled tube.

The pH of the product/LAL mixture at the compatible dilution/concentration was adjusted

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 sample volume. All tubes were placed in a non-circulating water bath at 37 1C
for 60  2 minutes. After the incubation period, each tube was carefully removed, one at a time, and
inverted slowly towards 180.

Phase II (Inhibition Enhancement / Validation Study):

A standard curve of CSE or RSE in LAL reagent water was prepared as For each batch to be tested
(3 batches were required for a full validation); A 6 mL sample at twice the dilution (Non-Interfering
Dilution) that was previously determined in the interference screen was prepared.

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A standard curve in each batch of product was prepared as follows:

1. 1ml sample at 2 x test dilution + 1ml LRW = negative control. NPC
2. 1ml sample at 2 x test dilution + 1ml 4 = test dilution at 2.
3. 1ml sample at 2 x test dilution + 1ml 2 = test dilution at.
PPC
4. 1ml sample at 2 x test dilution + 1ml  = test dilution at ½.
5. 1ml sample at 2 x test dilution + 1ml ½ = test dilution at ¼.

100 µL of each test specimen and each concentration of standard in water were inoculated into
each of 4 depyrogenated LAL grade tubes to prepare positive product control (PPC) and positive water
control (PWC) respectively, 100µL of compatible dilution of product and LRW was transferred into each
of 4 reaction tubes to prepare negative product control (NPC) and water negative controls (NWC)
respectively.
100µL of reconstituted LAL reagent was added to each labeled tube. The pH of the product/LAL
mixture at the compatible dilution/concentration was adjusted between 6.5 and 8.0. All tubes were
placed in a non-circulating water bath at 37 1C for 60  2 minutes. After the incubation period, each
tube was carefully removed and inverted slowly towards 180 The results were valid if all tubes
containing negative controls were without gels and endpoint of the standard curves in water and product
were not greater than one twofold dilution either side of the lysate sensitivity and the endpoint of the
standard curve in the product is not greater than a twofold difference to the endpoint of the standard in
water. If the above criteria were met; the product had been validated for testing in the LAL gel clot assay.

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RESULTS

Test for Confirmation of Labeled LAL Reagent Sensitivity (0.03125 EU/mL):

The geometric mean (GM) endpoint concentration of the solutions was determined for 2 lots
received. For the 2nd lot number (H0K356), the geometric mean (GM) endpoint 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:

Once a non-interfering dilution had been identified, Phase II (Inhibition Enhancement / Validation
Study) was performed as elaborated below; where two identical series of product dilutions (two fold
dilutions), one spiked with 2, and one left unspiked were prepared. The results of Phase I reveal the
non-interfering dilution (NID) of the product. The non-interfering dilution (NID) is the first set of
positive product control (PPC) that shows a gel. TABLE I showed enhancement up to 1:100 (MVD)

The following test dilutions were prepared by 1:10, 1:20, 1:40, 1:80 and 1:100 in LAL reagent
grade water after heating at 70-80°C for 10 minutes to coagulate the proteins followed by
centrifugation at 2000 rpm for 10 minutes to remove debris.

TABLE I: LAL Assay Results of Antisnake Venom after Heat Denaturation

Sample dilution undiluted 1:10 1:20 1:40 1:80 1:100

Unspiked (NPC) ++ ++ ++ ++ ++ ++

Spiked (PPC) ++ ++ ++ ++ ++ ++

- NPC and PPC is Negative Product Control and Positive Product Control; respectively
- Negative Water Control (NWC) (LAL Water free of endotoxin) showed negative results and Positive
Water Control (PWC) (LAL Water spiked with 2λ endotoxin) showed positive ones.
- (--) stands for inhibition and (++) stands for enhancement.

The sample was analyzed using BD100 dispersing agent instead of LAL reagent grade water to
dissociate and disperse endotoxin molecules from complex biological products and vortexed
vigorously for at least 5 minutes between each dilution to prepare 1:10, 1:20, 1:40, 1:80 and 1:100
followed by centrifugation at 2000 rpm for 10 minutes to remove debris as presented in TABLE II.

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TABLE II: LAL Assay Results of Antisnake Venom after dilution with BD100 dispersing agent

Sample dilution undiluted 1:10 1:20 1:40 1:80 1:100

Unspiked (NPC) ++ ++ ++ ++ ++ ++

Spiked (PPC) ++ ++ ++ ++ ++ ++

- NPC and PPC is Negative Product Control and Positive Product Control; respectively.
- Negative Water Control (NWC) (LAL Water free of endotoxin) showed negative results and Positive
Water Control (PWC) (LAL Water spiked with 2λ endotoxin) showed positive ones.
- (--) stands for inhibition and (++) stands for enhancement.

TABLE III: LAL Assay Results of not Heat-activated Antisnake Venom after rehydration of
LAL Reagent with Endotoxin-Specific Buffer Solution

Sample dilution undiluted 1:10 1:20 1:40 1:80 1:100

Unspiked (NPC) ++ ++ ++ ++ ++ ++

Spiked (PPC) ++ ++ ++ ++ ++ ++

- NPC and PPC is Negative Product Control and Positive Product Control; respectively.
- Negative Water Control (NWC) (LAL Water free of endotoxin) showed negative results and Positive
Water Control (PWC) (LAL Water spiked with 2λ endotoxin) showed positive ones.
- (--) stands for inhibition and (++) stands for enhancement.

TABLE II & III showed enhancement up to 1:100 (MVD). In order to sort out this
inhibition/enhancement problem, the sample was heat-activated at 70-80°C for 10 minutes to
coagulate the proteins. In addition, the Endosafe LAL reagent was rehydrated with Endotoxin-Specific
Buffer Solution (BG-120) to block β-D-Glucans & LAL-Reactive Material (LAL-RM) as presented in
TABLE IV.

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TABLE IV: LAL Assay Results of Heat-activated Antisnake Venom and rehydration of LAL
Reagent with Endotoxin-Specific Buffer Solution

Sample dilution undiluted 1:10 1:20 1:40 1:80 1:100

Unspiked (NPC) ++ ++ ++ -- --- --

Spiked (PPC) ++ ++ ++ ++ ++ ++

- NPC and PPC is Negative Product Control and Positive Product Control; respectively.
- Negative Water Control (NWC) (LAL Water free of endotoxin) showed negative results and Positive
Water Control (PWC) (LAL Water spiked with 2λ endotoxin) showed positive ones.
- (--) stands for inhibition and (++) stands for enhancement.
TABLE IV shows enhancement up to 1:20 (MVD). Therefore the non-interfering dilution (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 non-interfering dilution had been identified, Phase II (Inhibition Enhancement /
Validation Study) was performed.

Phase II: (Inhibition Enhancement / Validation Study):

Two identical series of endotoxin dilutions bracketing , one prepared in LAL reagent grade
water and another prepared in product diluted to 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.

TABLE V: Labeled LAL Reagent Sensitivity for Sample Matrix (Endotoxin/product)

Negative
2  /2 /4
Replicates

Control
Endpoint Log endpoint Mean of logs
0.0625 0.03125 0.0156 0.0078
LRW
Eu/mL Eu/mL Eu/mL Eu/mL

1 + + - - - 0.03125 -1.522

2 + + - - - 0.03125 -1.522
-1.36
3 + - - - - 0.0625 -1.204

4 + - - - - 0.0625 -1.204

GM = antilog (Σe/f) = 0.0433 EU/mL.

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TABLE VI: Labeled LAL Reagent sensitivity for Water Matrix (Endotoxin/LAL Reagent
Water)
Negative
2  /2 /4
Replicates

control
Endpoint Log endpoint Mean of logs
0.0625 0.03125 0.0156 0.0078
LRW
Eu/mL Eu/mL Eu/mL Eu/mL

1 + + - - - 0.03125 -1.522

2 + + - - - 0.03125 -1.522
-1.44
3 + - - - - 0.0625 -1.204

4 + + - - - 0.03125 -1.522

GM = antilog (Σe/f) = 0.036 EU/mL.

The validation test results showed in TABLES V and VI were considered valid because the gel
endpoint values for each series of control standard endotoxin (CSE) dilutions in water and control
standard endotoxin (CSE) dilutions in product are within a twofold dilution of each other and a two-
fold dilution of the LAL claimed 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 had been validated for testing in the LAL gel clot assay and validation is conducted at this
dilution on two other batches of product which was nearly showed the same results.

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Discussion

Snakebite is a major socio-medical problem affecting many communities globally, especially

African and Asian countries. It is frequently treated with parenteral administration of horse or sheep-
derived antivenins aiming at neutralization of toxins. Currently, serum-based antivenin is the only
medically approved antidote against snakebite envenomation. It is usually pepsin refined F (ab)
fragments of IgG purified from the serum of a horse or sheep that has been immunized with the venom
of one or more species of snakes (23).

Our polyvalent antisnake was produced by immunizing individual horses with venom of a single
species (Cerastes cerastes, Naja haje (cobra), and Naja nigricollis) and mixing various hyper immune
plasma for fractionation and formulation were performed.

These polyspecific antivenins should be promoted whenever feasible technically, as they offer
clinical advantages like better usefulness than monospecific antivenins. Their use reduces the need for
identification of snakes prior to initiation of antivenom therapy and simplicity in logistics provides
great advantages (24).

Gram negative bacterial outer membrane Lipopolysaccharide (LPS) induces a cascade of defense
mechanism that is known as fever and inflammation. Endotoxins are negatively charged
macromolecules as small as 20-30 kDa, varying in size due to bacterial origin, the presence of divalent
cations or biological detergents. Bacterial endotoxin is the significant pyrogen that has been identified
as a contaminant in parenteral products (22), so it is necessary to determine and control the
contaminated endotoxin levels in injections as a quality-control measure (25).

Limulus amebocyte lysate (LAL) test is an alternative method to the rabbit pyrogen test that
focused on detection of pyrogenic substances in sterile parenteral drugs. Despite that, interferences
might face pharmacists working in pharmaceutical factories when using LAL test to detect the
endotoxin levels in sterile parenteral products. Such interferences might be inhibitory or false positive
interference (26). Inhibition is caused by any material known to denature protein or to inhibit enzyme
action; which can be over-come 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 inhibition or activation basically involve the use of
positive controls. Product samples are “Spiked” with known Endotoxin levels, preferably the same
levels used in standards prepared for sensitivity determinations. The endpoint detection for the product
sample should not be different from the end-point for the standard series (17).

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To adequately and properly test polyvalent snake venom antiserum samples by the LAL method,
the samples must not inhibit or enhance the gelation response or 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 LAL test using 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 the two lots of LAL-CSE. The test results
were shown to be valid as the lowest concentration of the standard solution (0.25) showed a negative
result in all replicate tests and the geometric mean end-point concentration was the measured
sensitivity of Lysate solution (EU/mL) for the two lots, so the labeled sensitivity 0.03125 EU/mL was
confirmed with this lysate; Two phases (phase I and phase II) of dilutions for the sample were
performed. Phase I was decided on a series of tenfold serial dilutions of the product. On the other side,
phase II was performed via two serial dilutions of the sample to more precisely determine the dilution
at which the interference is overcomed.

To avoid interfering test conditions, the USP allows drug product dilutions based on the
established endotoxin limits (18). Maximum Valid Dilution was calculated using the equation MVD=
(E.L x Conc. 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 TABLE I, II, 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 reactive material in plasma and not from endotoxin
contamination as the sample is endotoxin free (treatment with affinity chromatography). Finally, in
order to sort out this enhancement problem, the activated sample was heated at 70-80°C for 10 minutes
with rehydration of the Endosafe LAL reagent in Endotoxin-Specific Buffer Solution (BG-120) to
block β-D-Glucans and LAL-Reactive Material (LAL-RM). These findings nearly agree with
Pennamareddy et al., (14) who stated that heat denaturation was effective in solving interference
problem in biotherapeutic drugs.

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Poole 1991 (27) stated that Erythropoietin and liver albumin injections give false positive results
due to 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.

Intravenous immunoglobulin treatment has been found to produce false positive LAL test result.
Increasing the amounts of administered immunoglobulins increased the levels of LAL reactive
material in plasma. Amebocyte lysate contains substances called (1-3) beta-D-glucan sensitive factors
(28). These factors can activate LAL to produce false positive results for the presence of Endotoxin.
Person and Weary (29) addressed these false positive reactions caused by non Endotoxin substances.
Another accepted procedure by Donovan (19) for sera is the exposure of the sample to boiling
water for 2 or 3 minutes prior to further dilution to at least ten-fold. Also, Cooper (17) mentioned that
a biological product which contains a serine protease is a LAL reactive under certain conditions. Heat
treatment to denature the enzymes prior to 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 non-pyrogenic substances (30).

Phase I results revealed that the non-interfering dilution (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 measurements were 7 as
shown in TABLE V and VI. Therefore, there is no possibility of false positive results and the same
methodology could be applied for similar kind of products.

Conclusions

We are looking forward that this study will help pharmacists in biological factories to overcome
the problems they might face them during performing LAL test for parentral administration. It was
investigated that Heat-activation at 70-80°C for 10 minutes with rehydration of LAL reagent with
Endotoxin-Specific Buffer Solution is the best done to sort out the enhancement problem. Also, it was
noted that use of a more sensitive LAL reagent or test method increases the scope of dilution (i.e the
maximum valid dilution increases)

Acknowledgments

The authors would like to thank Dr. Nabil El Biblawy, Chairman and C.E.O of Egyptian
Company for Production of Vaccines, Sera & Drugs (EGYVAC) for his tremendous support during
this work.

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Conflict of Interest Statement

The authors declare that they have no conflict of interest. All authors read and approved the final
manuscript.

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