BET Newsletter A Charles River India Private Limited Communication
www.criverindia.com
Dear Reader,
I hope that you would have found our attempt to address the issues
related to Inhibition encountered during LAL testing useful. In
continuation of the same, I wish to bring to you the next part of the
newsletter devoted to the complex issue of Enhancement.
The phenomenon of enhancement is probably the most complex and
intriguing topic for anyone analyzing bacterial endotoxins by the
Limulus Amebocyte Lysate test. The alternative pathway that
works through the Factor G is the cause for the phenomenon of
enhancement. Factor G is a 'biosensor' which interacts with
'glucans' or 'LAL Reactive Material' and in turn activates the
'enzymatic cascade' to form a gel, which is the root cause for a lot
of debate and discussion.
The topic, therefore, is exhaustive and has many significant
implications viz. causing the 'false positives', thus making it very
important to delve on the relevant aspects at length.
We have made an attempt to provide an insight with a view to
design a strategy to overcome this interference caused by the
enhancement and enable successful LAL testing.
You are always welcome to send your suggestions and comments to
our email id : india.customercare@crl.com
Sincerely,
Dr. P. K. Chitnis
Director
Volume 1, Issue 5 : November 2010
Part II: Enhancement
Dr. K. Nagarajan and Dr. P. K. Chitnis
Introduction
Limulus Amebocyte Lysate (LAL) test is the assay of choice to
detect endotoxin because of its sensitivity and specificity
towards bacterial endotoxins {Lipopolysaccharide (LPS) from
the gram negative bacterial cell wall}1,2. However, a few
polymeric forms of glucose namely glucans have been shown to
activate the LAL cascade of enzymes when they are present in
sufficient quantities in the sample solution3. The glucans
include -(1,3)-D-glucans, which are found in the cell wall of
fungi, yeasts and alage4,5 and LAL-reactive material (LAL-RM)
presumably -(1,4)-D-glucan derived from a hollow-fiber
hemodialyzer with a saponified cellulose acetate or Cuprophan
membrane6. It is very interesting to note that not all of the
polymeric forms of glucose react with the LAL. A better
understanding of this phenomenon of non-reactivity of some
types of glucans to LAL relates back to the principles of the LAL
test itself.
The clotting mechanism of the blood of the horseshoe crab is
designed to prevent the spread of bacterial/fungal
contamination throughout the circulatory system of the
horseshoe crab7. When the LPS of Gram-negative bacteria/-
(1,3)-D-glucans come in contact with the blood cells of the
horseshoe crab an enzymatic cascade is triggered (Figure.1).
The reaction pathway consists of two biosensors, Factor C and
Factor G, two proenzymes (Factor B and proclotting enzyme)
and a clottable protein (coagulogen). This enzymatic cascade
forms the defense mechanism of the horseshoe crab aimed at
detecting bacterial endotoxins with the help of Factor C and
glucans with the help of Factor G. The two pathways merge at
the activation step of the proclotting enzyme (Figure 1)7.
Figure 2. Cascade mechanism of LAL with endotoxin and -glucans
In order to gain a better understanding of the two cascades
induced by the endotoxin and glucan, it is important to look at
the glucans and LAL reactive materials in greater detail.
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BET Newsletter : A Charles River India Private Limited Communication Volume 1, Issue 5 : November 2010

-(1,3) Glucans8
(1,3) Glucans are those polymers of D-glucose linked
glycosidically with a -(1,3) linkage (Figure 2).
Depending upon the concentration and size of the glucans,
they might interfere in the LAL testing by causing the formation
of a gel clot in the absence of endotoxin. This gelation in
absence of endotoxin is contrary to the expected result and is
therefore referred to as a False Positive.

Novitsky et.al, studied10 the mechanism of LAL with endotoxin

and -glucans in detail and his observation as follows:
(a)
1. LAL reagent more sensitive to endotoxins than glucans or LAL-RM.

2. The conformation and degree of polymerization of the glucose

molecule represents a potential source of variability for LAL
reactivity. The reactivity of LAL reagent to glucan molecule
depends upon molecular weight, the degree of branching and the
(b) conformation of the glucan. A high molecular weights (1,3)-D-
glucans were more reactive than lower molecular weights, single
stranded glucans were more active than those of helical
structure11.

3. LAL formulated with Zwittergent exhibits decreased sensitivity to

LAL-RM and increased sensitivity to Endotoxins.

Figure 2. (a) Glucose molecule showing carbon numbering notation and - orientation 4. Chloroform extraction of LAL increased sensitivity of the reagent to
(b) Showing orientation and location of different -glucan linkage endotoxin, lipid A and LAL-RM/Glucans.

Examples of -Glucans An independent study12 by Koch et al. showed that greater

variations occur in quantifying glucans than in quantifying
Name Glycosidic linkage endotoxins. Further to this, he observed that the glucan
Cellulose -1,4 pathway is totally blocked by laminarin at certain
Curdlan -1,3 concentrations without affecting the endotoxin activated path,
thereby allowing endotoxins to be quantified by the LAL test.
Laminarin -1,3 & -1,4
Chrysolaminarin -1,3
M. Ochiai et al. during their study13 of the applicability of the
Lentinan -1,6: -1,3 endotoxin test (BET) to IHA [Influenza HA] vaccine, they
Lichenin -1,3 & -1,4 observed that most of the batches of vaccine showed
Pleuran -1,3 & -1,6 considerable LAL activity inspite of lacking pyrogenicity in
Zymosan -1,3 rabbits. The LAL activity of IHA vaccine showed different
physiochemical properties from those of LAL activity of
endotoxins. It was also observed that the LAL activity of
LAL Reactive Material9,5 endotoxin is known to be sensitive to polymyxin B treatment and
LAL reactive material (LAL-RM) is released into the filtrate by was found to be resistant to polyoxyethylene 10 cetyl ether
hollow fiber filters made of some types of derivatized cellulose (Brij56) treatment.
acetate. This problem is most immediate in kidney dialysis
clinics using these kinds of filters. LAL-RM ranges in molecular
S. Tanaka et al. carried out an extensive study14 on the effects of
weight from 5000 to 70,000 daltons and has an average
various -D-glucans on the coagulation cascade in horseshoe
molecular weight of 24,000.
crab amebocyte lysate and reported that activation of Factor G
required (1,3)--D-glycosidic linkages and was influenced by
Sources of Glucan and LAL-reactive material
molecular weight, configuration and concentration of -glucans
Glucan contamination will arise within pharmaceutical
[Figure 3]. Relatively low molecular weight laminarin
processing, either from fungal contamination (or yeast
oligosaccharides and curdlan degradation products
hydrolysate) of the process OR from using certain products
specifically inhibit the factor G pathway at certain
containing cellulose or chitin. The microbial contaminants
concentrations. Hence soluble inhibitors from this class are
include zymosan (from yeast), laminarin (from alage), lentinan
useful in the preparation of endotoxin-specific LAL reagents.
(from fungi) and curdlan (from bacteria)6. The cellulosic glucan
origin arises from cellulosic filters and cotton wool.

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BET Newsletter : A Charles River India Private Limited Communication Volume 1, Issue 5 : November 2010

Flow Chart for Glucan/LAL-RM

Uncharacterized
Drug Product

Preliminary Testing

Results

Unspiked sample: + + at MVD/MVC

Spiked Sample : + + at MVD/MVC

Figure 3. Effect of Concentration of (1,3)-D-glucans having different number Product fails

average molecular weight on their property to activate factor G.

Test at 2 fold dilutions

beyond MVD

Unspiked sample: -- at 2MVD or 4MVD -- at 8MVD or more

Spiked Sample : + + at 2MVD or 4MVD + + at 8MVD or more

Endotoxin contamination LAL-RM/Glucans or High

at borderline level Endotoxin contamination

Reduce in-process Use a Glucan-blocking

pyroburden buffer and test at MVD

Fails at MVD Passes at MVD

Figure 4. Conventional LAL response to Glucan and Endotoxin (Endotoxin

and Glucan react synergistically to LAL. LAL-RM/Glucans
Endotoxin contamination
contamination

A soluble, carboxymethylated curdlan (CM-curdlan) developed

Reduce in-process Routine testing with
as a -glucan blocker for LAL reagents, activates LAL in the pyroburden Glucan-blocking buffer

range of 1-1000ng/mL, but is inactive in concentrations above

0.1ng/mL and blocks other LAL-active glucans in this range15.
The following case studies would further explain the approach
mentioned above:
When Mr. T.E. Munson, Chief, Sterile Drugs Branch, USFDA was
asked about the glucan contamination observed in Case Study15
pharmaceuticals and its effect on the LAL reaction, he replied Example 1
as follows Glucans and LAL-RM do not appear to be a problem Method: Kinetic Chromogenic Assay
in pharmaceuticals. I have not heard or seen any data that
Product: Antibiotic bulk
indicates that a pharmaceutical was contaminated with glucans
Standard Curve: 5-0.05 EU/mL
or LAL-RM. I have also not seen any data or reports that indicate
Endotoxin Limit: 0.1 EU/mg
that glucans or LAL-RM is toxic in the doses that would be present
Minimum Valid concentration (MVC): 0.5 mg/mL
if they were contaminants. With respect to LAL-RM, it has only
been isolated with cellulose-based membranes in Test Concentration: 3.6 mg/mL
hemodialyzers. There is no data to date that conclusively links Spike concentration: 0.5 EU/mL
LAL-RM to any adverse reactions seen in hemodialysis patients.
Result
Presence of trace of LAL-reactive Glucan present in test sample gives
Management of Glucans while testing for Bacterials
puzzling results and table 1 presents the findings of KCA on 3 lots of
Endotoxins in Pharmaceutical preparations bulk antibiotic.
In order to differentiate the glucan and endotoxin activation of
LAL, tests are conducted parallel with and without glucan Without LRG Blocker With LRG Blocker
Lot EU/mg Recovery (%) EU/mg Recovery (%)
blocker. A Flow chart below summarizes the sequence of LAL
A <0.14 110 <0.14 90
testing to differentiate Glucan and Endotoxin by gel-clot
B <0.14 152 <0.14 82
method16. C 0.1 337 <0.14 99

Table 1. Effects of Low-level LAL-reactive Glucan (LRG) contamination

on Kinetic Chromogenic LAL analyses of bulk antibiotic.

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BET Newsletter : A Charles River India Private Limited Communication Volume 1, Issue 5 : November 2010

Observation Each cartridge contains a precise amount of glucan specific

Lot A of the bulk antibiotic had no detectable endotoxin in the LAL formulation, chromogenic substrate and a glucan
range of standard curve with both glucan-blocked and glucan- standard.
sensitive LAL reagents.
The glucan cartridges have a sensitivity range of 10-1,000
Lot B of the bulk antibiotic has no detectable endotoxin in the pg/mL and yield results in less than 30 minutes.
range of standard curve with both glucan-blocked and glucan
sensitive LAL reagents. The recovery has shown variation The Glucan tests can be run on the same Endosafe PTS reader
with/without glucan blocker. that is used to detect endotoxin.
Endotoxin
Lot C yielded endotoxin value equal to endotoxin limit with
Factor C Factor C(active) (1,3)--D-Glucan
invalid spike recovery i.e. 337% (Acceptable range is 50-200%)
without glucan blocker while using glucan blocker it yielded no Factor B Factor B (active) Factor G Factor G (active)
detectable endotoxin in the range of standard curve with valid
Proclotting Enzyme Clotting Enzyme
spike recovery i.e. 99%.
Substrate-p-nitroaniline p-nitroaniline
Inference
The study revealed advantages of using kinetic LAL method Figure 5. Limulus Enzyme Cascade

over gel-clot to test glucan suspected product because

endotoxin and glucan react synergistically in LAL.
Example 2
The characteristics of the LAL reactive substance (LRS) in
Sandostatin and CM-cellulose (CMC) were investigated in detail
by M. Tsuchiya and made the following observations17:
Cartridge Reagents
Each Endosafe-PTS cartridge contains four channels to which
glucan specific LAL reagent and a chromogenic substrate have
been applied. Two of the four channels also contain a glucan
spike and serves as the positive product controls (Figure. 6.)

a) The LAL reactive substance in Sandostatin and CM-cellulose is not

pyrogenic.

b) The characteristics of the LAL reactive substance are different from

those of endotoxin.

c) The reactivity of the LAL reactive substance in Sandostatin and CM-

cellulose can be eliminated by the use of 2 % NaCl or use of Tris-GB
(Glucan blocking) buffer as a diluent of the samples.

d) The use of Tris-GB buffer will be the best solution for the BET for
Sandostatin and CM-cellulose related product.

e) Best recommended method for the BET of Sandostatin and CMC
related product is as below:
1) Sample: 10-fold dilution with Tris-GB buffer
2) Method: KTA with the microplate method
Rapid method to Detect of Glucan using Portable Test
System (PTS)
Charles River Laboratories offer a new test to help ensure that
the products are as free of glucans. The Endosafe-PTS glucan
assay is a rapid test designed to detect and quantify (1,3)- -D-
Glucans in a sample.
This test employs the Limulus Enzyme Cascade to detect (1,3)-
-D-Glucan (Figure.5.) in the sample. The Endosafe-PTS
cartridge and its interface with the reader have been designed
on the principles of the Kinetic Chromogenic (KCA) method to
measure the color intensity which is directly related to the (1,3)-
-D-Glucan concentration in a sample.
Figure 6. Glucan Cartridge Setup
Conclusion
It would be most appropriate, therefore, to surmise that
Enhancement is more of a Glucan Interference to LAL testing
than anything else.
Further to this, considering the fact that the coagulation system
of the Horseshoe crab is activated by two pathways viz. by
endotoxin (LPS) and by (1,3)--D-Glucans, a differentiation
between the two can be achieved based on their reactivity
towards LAL reagent. Thus, glucan interference of LAL test can
be managed in two ways:
1. The first approach combines the use of glucan sensitive and glucan
blocked LAL to differentiate endotoxin and -glucan and to identify sources
of glucan contamination with the aim of eliminating them from the product
and,
2. The second approach uses glucan-blocked reagents for release of testing
of certain products, such as medical devices and carboxymethylated
polysaccharides, where the objective is to test for endotoxin specifically.

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BET Newsletter : A Charles River India Private Limited Communication Volume 1, Issue 5 : November 2010

References Events and Happenings

1. J. Levin and F. B. Bang, Bull. Johns Hopkins Hosp. 115, 265-274 (1964).
Kinetic LAL Workshop at Hyderabad
2. J. Levin and F. B. Bang, Thromb. Diath. Haemorrh. 19, 186-197 (1968). A Kinetic LAL Workshop was held on the 24th & 25th of
September 2010 at Hotel Marriott, Hyderabad.
3. (a) A. Kainuma, T. Asano, H. Torii and Y. Sugino, Biochem. Biophys. Res.
Commun., 101, 434-439 (1981). (a) S. Bartnicki-Garcia, Proceedings of the
Phytochemical Society symposium1969, Academic Press, London, Page
no.81-103 (1970). (b) K. Ikegami, K. Ikemura, T. Shimazu, M. Shibuya, H.
Sugimoto, T. Yoshioka and T. Sugimoto, J. Trauma, 28, 1118-1126 (1988). (c)
K. Ikemura, K. Ikegami, T. Shimazu, T. Yoshioka and T. Sugimoto, J. Clin.
Microbiol. 27, 1965-1968 (1989).

4. A. Kakinuma, T. Asano, H. Toril and Y. Sugino, Biochem. Biophys. Res.

Commun. 101, 434-439 (1981).

5. (a) F. C. Pearson, J. Bobon, W. Lee, G. Brusaer, M. Sagoma, G. Jakubowski,

R. Dawe, D. Morrison and C. Dinarello. Artif. Organs, 8, 291-298 (1984).
(b) L. A. Carson and J. Petersen, In S. W. Watson, J. Levin and T. J. Novitsky
(ed), Alan. R. Liss. Inc., New York, Page No: 217-230 (1982).

6. T. Sandle, Pharmaceutical Microbiology Blog, 2010.

7. T. Mortia, S. Tanaka, T. Nakamura and S. Iwanaga, FEBS Lett. 129, 318-321

(1981).

8. H. A. Harper, V. W. Rodwell and P. A. Mayes, Review of Physiological

Chemistry, 17th Edition, 1979.
Kinetic LAL Workshop at Indore
9. F. C. Pearson, Blood Purification, 5, 115-122 (1987).
Another Kinetic LAL Workshop was conducted successfully on
10. P. F. Roslansky and T. J. Novitsky, Journal of Clinical Microbiology, 29, 2477- the 25th & 26th of November 2010 at Hotel Fortune Select
2483. Landmark, Indore.
11. N. Ohno, Y. Emori, T. Yadomae, K. Saito, A. Masuda and S. Oikawa,
Carbohydr. Res. 207, 311-318(1990).

12. G-H. Zhang, L. Baek, O. Buchardt and C. Koch, Journal of Clinical

Microbiology, 32, 1537-1541(1994).

13. M. Ochiai, H. Tamura, A. Yamamoto, M. Aizawa, M. Kataoka, H. Toyozumi and

Y. Horiuchi, Microbiol. Immunol., 46, 527-533 (2002).

14. S. Tanaka, J. Aketagawa, S. Takahashi, Y. Shibata, Y. Tsumaraya and Y.

Hashimoto, Carbohydr. Res. 244, 115-127 (1993).

15. (a) J. F. Cooper, M. E. Weary and F. T. Jordan, J. Parenter. Sci. Technol. 51, 2-
6 (1997) (b) J. F. Cooper, Journal of parenteral Science and Technology, Nr.
44:1 Page No: 13 (1990).

16. H. S. Kumar, LAL Newsletter (Salesworth India Pvt. Ltd.), 6, 1-6 (1998).

17. M. Tsuchiya, Charles River Laboratories, USA, Personnel Communication.

Contact Us
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