BET Newsletter

A Charles River India Private Limited Communication

Dear Reader,

Volume 1, Issue 4 : July 2010

www.criverindia.com

Inhibition and Enhancement

Dr. K. Nagarajan, Mr. Shibu Chakraborty and Dr. P. K. Chitnis

With the completion of the first quarter of our fiscal year and after
addressing some introductory issues, I wish to bring to you a couple
of technically oriented newsletters focused at addressing very
pertinent issues faced during analyses viz. the interference to the
LAL test.
The topic is exhaustive and has many ramifications, thus making it
imperative to address the relevant areas in two separate
newsletters.
The phenomenon of inhibition is by far the most widely observed
and also is probably the most widely studied. It is important to gain
an insight into the aspects of barriers to the successful completion
of the cascade and endotoxin depletion, the foci of the entire
gambit of inhibition. The endotoxin when is not available to the
LAL reagent, we do not observe gelation and refer to it as
inhibition. Over the years, beginning with the pioneering work of
Fredick Bang, Jack Levin and James Cooper, several factors
have been identified to contribute to inhibition. The process
continues today with the advent of new drug and drug technologies
which provide us with new challenges.
Therefore, an insight into these factors and their contributing
actions is imperative to design a strategy to overcome this
inhibition and pave the way for successful LAL testing.
I must say that Dr. Nagarajan, a chemist by education and
training, has added a chemist's viewpoint very nicely to this
newsletter.
You are always welcome to send your suggestions and comments to
our email id : india.customercare@crl.com
Sincerely,

Dr. P. K. Chitnis
Director

Introduction
The Limulus Amebocyte Lysate (LAL) test is widely used for
the determination of bacterial endotoxins in pharmaceutical
products and medical devices1. The test is based on the ability
of the endotoxin to activate an enzyme cascade present in the
amebocytes of the Limulus polyphemus2,3. In order to test
a pharmaceutical product for bacterial endotoxins by the
LAL test, the test method must first be validated for the product.
The procedure of Validation of the Product involves the
establishment of the fact that the sensitivity of the test method
to detect the endotoxin in a specific drug product using
pharmacopoeially accepted LAL methods is within acceptable
limits of interference.
It is a well established fact that while setting up a Standard
Operating Procedure (SOP) for a product to be tested by the
LAL/BET, it is imperative for one to establish the level of
interference encountered while testing the product and outline
an effective way to mitigate the interference. Guidelines to this
effect have been elaborately outlined in regulatory documents4,5
while making the product testable.
Interference to the LAL test can be broadly grouped as:
1. Inhibition factors that attenuate the enzymatic cascade
2. Enhancement factors that augment the enzymatic cascade
An FDA survey on LAL test compatibility with human drugs has
documented that only about 30% of products can be directly
tested6 while others show inhibition. Of these 97% of the
inhibitory problems are seen to be resolved by mere dilutions
thereby clearly indicating that the inhibitory conditions are due
to concentration of product rather than the test method itself.
The topic on interference is very exhaustive and needs to be
treated in detail so as to do justice to all the relevant aspects of
this phenomenon. The authors therefore have found it ideal to
deal with this complex and exhaustive topic in two separate
newsletters devoted to the two aspects of Interference viz.
Inhibition and Enhancement.

Part I: Inhibition
Part II: Enhancement

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Volume 1, Issue 4 : July 2010

BET Newsletter : A Charles River India Private Limited Communication

Part I: Inhibition
Inhibition Mechanism
When testing an undiluted product, the product usually suffers
from inhibition to the LAL test which is due to several factors
like7:
1. Suboptimal pH conditions
2. Aggregation or adsorption of control endotoxin spikes
3. Unsuitable cation concentrations
4. Container effects
5. Enzyme or protein modification
1. Suboptimal pH Conditions
As is well known that the enzyme activity is affected by pH
(i.e. chemical environment), enzymes usually have a fairly
narrow range of pH optimum for activity e.g. LAL endotoxin
reaction it is optimized in the range of 6-812. LAL reactivity is
markedly suppressed as pH is increased or decreased from
neutrality by altering enzyme conformation. [Note: As a part of
validation one should check the pH of the mixture of LAL
reagent and sample solution12].
Remedial Measures
1. The LAL reagent has been provided with a sufficient buffer
capacity to overcome minor pH problems. This capacity
markedly varies from one lysate formulation to another.
2. Taking advantage of the allowable dilution of a sample i.e.
Maximum Valid Dilution (MVD) which is allowed while
performing the LAL test, minor pH problems can be
overcome. It can be noted that dilution has a great effect on
the pH of a strong acid as compared to a weak acid.
3. For highly acidic or basic samples it is advisable to resort to
adjustment of the pH using either:
a. 0.1N or lower concentration of HCl or NaOH, or
b. Tris HCl buffer or Tris base buffer
In order to achieve best results, it has been recommended to
use Tris buffer (buffering range 7-9) for low pH products instead
of concentrated NaOH. Use of concentrated NaOH may cause
endotoxin inactivation in the product.
Use of lower concentration of NaOH and HCl (i.e. like 0.1 N)
involves preparation of solution using endotoxin free water
(LRW) and depyrogenated glassware.
2. Endotoxin Modification
Endotoxins are lipopolysaccharide molecules (LPS) found in
the cell wall of gram negative bacteria. LPS consists of a
polysaccharide chain (hydrophilic moiety) and a lipid A
(hydrophobic moiety). LPS is a complex amphilic molecule with
high molecular weight.

Page 2 of 5

The amphilic nature of LPS permits interaction between other

LPS moieties under the presence of certain test specimen
component like monovalent cations (like Na+) and polyvalent
anions (e.g. EDTA) which leads to the micelle formation13,14.
Generally, a large increase in ionic strength (i.e. ion
concentration) of a test specimen leads to molecular
aggregation, thereby leading to a poor recovery of purified
endotoxin, i.e. poor endotoxin recovery.
Remedial Measures
1. Sample dilution
2. Use of endotoxin dispersing agents like BD100*
3. Unbalanced Divalent Cation Levels
Divalent cations play an important role in endotoxin reactivity
and in the LAL reaction. The presence of negatively charged
LPS can bind to divalent cations leading to aggregation which
in turn reduces the endotoxin reactivity towards LAL reagent.
Divalent cation increases the aggregation between endotoxin
moieties through cation bridging15-17. Optimum levels of divalent
cation concentration are required for LAL reactivity but on the
other hand higher concentrations might lead to inhibition.
Presence of chelators like EDTA, ethylene diamine in the drug
product may cause insufficient availability of the cations due to
chelation effect. [Note: In the event that if Positive Product Control
(PPC) is found -ve (in Gel Clot Method) or if Spike recovery is below 50%
(in Kinetic Method), should one look into this option].

Remedial Measures
Use of Calcium Tris buffer and Magnesium Tris buffer to
overcome this problem.
4. Container Effects
It has been observed that, upon long periods of exposure
endotoxins are adsorbed on containers like polypropylene7,18
which causes poor recovery of endotoxins. To avoid adsorption
of endotoxins on containers either high quality borosilicate
glass or polystyrene tubes are used while performing LAL
assays. The presence of cationic protein like lysozyme, histone
affects the endotoxin recovery in glass tubes and the presence
of non-cationic protein like Bovine Serum Albumin (BSA) affects
the endotoxin recovery in polystyrene tubes by adsorption19,20. It
is therefore recommended that care must be taken in selecting
containers used during the LAL assay.
While reusing glassware, care must be taken because the
cleaning process involves washing the glassware with
detergents and chromic acid, as both are causes of endotoxin
depletion by micelle formation and endotoxin inactivation,
respectively. Thus, cleaning of glassware too can cause
problems in endotoxin recovery.
Unpurified Endotoxin - Lipopolysaccharide + Protein
Purified Endotoxin - Lipolpolysaccharide
* Please refer CRL Product Catalog No. BD100

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Volume 1, Issue 4 : July 2010

BET Newsletter : A Charles River India Private Limited Communication

5. Protein or Enzyme Modification

The phemomenon of gelation is caused due to an enzymatic
coagulation cascade that involves serine proteases. The
occurrence of inactivation of enzymes due to oxidants, antioxidants, proteolytic agents or specific inactivators causes
inhibition. Apart from diluting the sample other approaches
employed to resolve these problems include chemical or heat
inactivation of the inhibiting factor component or ultrafiltration
to separate endotoxin from the rest of the test specimen.
Problem

Gel Clot Method

Example
Product name : Diclofenac Sodium
Endotoxin Limit : 2.0 EU/mg
Lysate Sensitivity (l
) : 0.125 EU/mL

MVC =

Labeled Lysate Sensivity (l

)
Endotoxin Limit

Solution

Suboptimal pH

a. Simple dilution
b. Addition of dilute acid or base
c. Neutralize with Tris buffer

Endotoxin depletion

a. Simple dilution plus vortexing

b. Endotoxin dispersing agent

Container Effects

a. Avoid polypropylene tubes

b. Use of disposable polystyrene tube
is recommended

Calcium depletion

a. Dilute out chelation effect

b. Use of Ca Tris and Mg Tris buffers

Protein denaturation

a. Neutralize offending agent by heat

treatment

Table 1. Summarizes the type of inhibition and the corrective action

Screening for Inhibition

As mentioned earlier, the validation of a product entails a test
condition where an endotoxin standard is detected with the
same efficiency in a test sample as against a presumable
neutral milieu like LAL Reagent Water (LRW). Prior to validation
of a product, Preliminary screening or Inhibition-Enhancement
testing of the product should be carried out. This study helps
one to determine Non-Interfering Dilution (NID) or NonInterfering Concentration (NIC) where the product does not
show interference in the form of Inhibition or Enhancement$.

MVC =

0.125 EU/mL
2.0 EU/mg

= 0.0625 mg/mL

Sample Concentrations (mg/mL)

1.0

0.5

0.25

0.125

0.0625

Unspike sample (NPC) (in duplicate)

--

--

--

--

--

Spiked Sample (PPC) (2l

CSE)

--

++

++

++

++

This assay shows that inhibition at a test sample concentration

of 1.0 mg/mL since there was no spike recovery at this
concentration. Therefore, the non-interfering concentration
(NIC) is 0.5 mg/mL.
Preliminary Screening by Kinetic Turbidimetric
Method (KTA)
Example
Product name : Gentamycin 40 mg/mL
Endotoxin Limit : 0.71 EU/mg (USP)
Standard Curve : 5.0 - 0.05 EU/mL

MVC =
MVC =

Least Concentration of Endotoxin

Endotoxin Limit

0.05 EU/mL
0.71 EU/mg

= 0.070 mg/mL

Positive Product Control (PPC) is a serial two-fold dilution of

the product up to MVD or MVC21 when prepared with the
addition of 2l
concentration of CSE in case of Gel Clot method
OR concentration equal to the mid-point of the standard curve
in case of Kinetic Method. This addition of specified amount of
endotoxin is referred to as Product Spiking.
Spiking can be carried out by the following two methods:
1. Double Strength Method or 50:50 Method
2. Hot-Spike Method
$ Discussion restricted to Inhibition
Terms are used by Endosafe - Charles River Laboratories
and would not be found in any regulatory documents

Page 3 of 5

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Volume 1, Issue 4 : July 2010

BET Newsletter : A Charles River India Private Limited Communication

Flow Chart for Preliminary Testing

Uncharacterized
Drug Product

Preliminary Testing

Results

Unspiked sample:
Spiked Sample :

-- -- at MVD/MVC
+ + at MVD/MVC

-- -- at MVD/MVC
-- at MVD/MVC

+ + at MVD/MVC
+ + at MVD/MVC

Validation at a
Dilution/Concentration between
MVD/MVC and NID/NIC

Use a more
sensitive lysate

Product fails

Routine testing

Repeat
preliminary testing

Validity of KTA test result by regulatory document

a. The absolute value of the correlation coefficient, |r|, must
be greater than or equal to 0.98, i.e. r 0.98, for the range
of endotoxin concentrations indicated by the Lysate
manufacturer.
b. Reaction time of water control should be greater that of
onset OD reaction time of least concentration of endotoxin
of standard curve.

Conclusion
Thus, a rational approach to solving the inhibition problems
would be to understand the nature of interference encountered
and accordingly adopting a procedure to resolve the problem
by using either the most preferred approach of dilution or
pretreatment of samples before subjecting to LAL testing.

c. Spike recovery in the range of 50% to 200%.

Glossary
Sample Concentrations (mg/mL)
NPC (EU/mg)
Spike Recovery (%)

1.0

0.5

0.25

0.125

<0.05

<0.1

<0.2

<0.4

30

63

83

84

In this context before venturing into the details of the mechanism of action of
the inhibiting factors, we would like to start with the basics in order to help
resolve the problem caused by the above mentioned inhibiting factors.
pH
It is a measure of acidity or basicity of a solution. By definition, the negative
logarithm (base10) of concentration of hydronium ions (H3O+).
pH = -log10{[H+]}

This assay shows that inhibition is encountered at a test sample

concentration of 1.0 mg/mL which is indicated by the spike
recovery of 30% which is below the acceptable lower limits of
50%.

Note: In real solutions not concentrations [H+], but ion activities (aH+) should
be used for calculations8,9.
pH = -log (aH+)
In diluted solutions, aH+ = [H+], thus

Further to this, it can be observed that the spike recovered is

falling within the acceptable range of 50-200% at concentration
ranging from 0.5 to 0.125mg/mL. Hence, non-interfering
concentration (NIC) is 0.5mg/mL
A flow chart below helps to summarize the sequence of LAL
testing for an uncharacterized drug product 22.

pH = -log10{[H+]}
A low pH indicates a high concentration of hydronium ions while a high pH
indicates a low concentration of hydronium ions.
A buffering solution is required to adjust the adverse pH conditions of the
sample solution because of LAL - test inhibition. The function of a buffering
agent/solution is to drive an acidic or basic solution to a certain pH state and
the definition of buffer solution is:
Buffer Solution
It is an aqueous solution consisting of a mixture of a weak acid and its
conjugate base or a weak base and its conjugate acid. The property of this
buffer solution is that its pH changes very little when a small amount of strong
acid or base is added to it. Buffer solutions are used as a means of
maintaining the pH at a nearly constant value in a wide variety of chemical
applications.

Page 4 of 5

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Volume 1, Issue 4 : July 2010

BET Newsletter : A Charles River India Private Limited Communication

Example
Tris Buffer Tris is an abbreviation of the organic compound known as Tris
(hydroxymethyl) amino methane with the formula {(HOCH2)3CNH2}. The
buffering range for Tris is 7-9.
Tris HCl Buffer A common variant of Tris (i.e. tris base) is Tris-HCl, the acid salt.
When titrated to a specific pH with the corresponding counter ion (OH- for TrisHCl, H+ for Tris base) with the formula {(HOCH2)3CNH2}.HCl.
Micelle
It is an aggregate of surfactant molecules dispersed in a liquid colloid. A
typical micelle in aqueous solution forms an aggregate with the hydrophilic
head regions in contact with surrounding solvent, sequestering the
hydrophobic single tail region in the micelle centre.

Figure 1. Micelle - The lipophilic tails of the surfactant molecule remain on the
inside of the micelle due to unfavourable interactions. The polar head of the
10
micelle due to favourable interations with water form a hydrophilic outer layer .

Ionic Strength
It is the measure of concentrations of ions in the solution. Ionic compound
when dissolved in water, dissociates into ions. One of the main characteristics
of a solution with dissolved ions is the ionic strength.

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

Chemistry, 17th Edition, 1979.
11. J. E. Huheey, E. A. Keiter and R. L. Keiter, Inorganic Chemistry, 4th Edition.
12. USP convention Pharmacopeial Forum 35, 699-707 (2009); J. F. Cooper,
Charles River Endosafe LAL Times, Vol. 4, No.2, 1-4 (1997).
13. A. Bergstrand, C. Svanberg, M. Langton and M. Nyden, Colloids and
Surfaces B: Biointerfaces, 53, 9-14 (2006).
14. D. Stiger, J. Phys. Chem., 78, 2480-2485(1974).
15. J. G. Lessard and M. Fragata J. Phys. Chem. 90, 811-817 (1986).
16. E. Rowatt and R. J. P. Williams, Biochem. J. 245, 641-647 (1987).
17. A. M. Field, E. Rowatt and R. J. P. Williams, Biochem. J. 263, 695-702
(1989).
18. T. J. Novitsky, J. Schmidt-Gengenbach and J. F. Remillard, J. Parenter, Sci.
Technol. 40, 284-286(1986).
19. H. Yokota, H. Kiyonasa, H. Kaniwa and N. Saisho, J. Pharm. Biomed. Anal.
22, 757-761(2000).
20. H. Yokota, H. Kiyonasa, H. Kaniwa and T. Shibanuma, J. Pharm. Biomed.
Anal. 25, 1001-07(2001).
21. K. Nagarajan and P. K. Chitnis., BET Newsletter (A Charles River India
Private Limited Communication), Volume No: 1, Issue No: 3, 1-5 (2010) and
reference cited therein.
22. H.S. Kumar, LAL Newsletter (A Salesworth India Pvt. Ltd., India) 6, 1-6
(1998).

Events and Happenings

Endotoxin Seminar
A seminar on Rapid Endotoxin Detection Systems was held
on 11th of June 2010 at Fortune Select Palms, Chennai.

Chelation
It is a phenomenon of formation of two or more separate coordination binding
between bidentate or a polydentate ligand and a single central atom11. Usually
these ligands are organic compounds and are called chelators. E.g.: Ethylene
diamine, Ethylene Diamine Tetra Acetate (EDTA)

Figure 2. Ethylene diamine tetra acetate binding to a central metal ion with six bonds11

References
1.
2.
3.
4.

5.
6.
7.
8.
9.

J.F. Cooper and M.E. Neely, Pharm. Technol. 4, 72-79 (1980).

J. Levin and F. B. Bang, Thromb. Diath. Haemorth., 19, 186-197 (1968).
K. Duner, J. Biochem. Biophys. Meth. 26, 131-142(1993).
Guideline on validation of the LAL test as an end-product endotoxin test for
human and animal parenteral drugs, biological products and medical
devices, Food and Drug Adm., Dec. 1987.
USP 28-NF23 Supplement 2 Chapter on <85> Bacterial Endotoxins Test.
C. W. Twohy, A. P. Duran, T. E. Munson and J. Parenter, Sci. Technol. 30,
190(1984).
J. F. Cooper, J. Parenter, Sci. Technol., 44, 13-15 (1990).
P. W. Atkins, The Elements of Physical Chemistry, 3rd Edition.
A. K. Covington, R. G. Bates and R. A. Durst, Pure & Appl. Chem., 57, 531-

Page 5 of 5

Whats Next?
The next Kinetic LAL Workshop to take place at Hyderabad in
September 2010. The details will be announced shortly.

Contact Us
Charles River Laboratories India Private Limited
907, Barton Centre, 84, M.G. Road, Bangalore 560 001
Tel: 080-25588175 - 177. Fax: 080-41659281. Email: india.customercare@crl.com
Mumbai Tel: 022-25905203. Fax: 022-25911464. Email: bbyoffice@crl.com
Ahmedabad Tel: 079-26407713. Fax: 079-26406992. Email: ahdoffice@crl.com
Hyderabad Telefax: 040-27177710. Email: hydoffice@crl.com

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