Pyrogen testing 112070804005

GUIDED BY : PRESENTED BY :
Mr. Anand K. Patel PARTH PATEL
M.Pharm Sem-1
Roll no: 5

DEPARTMENT OF QUALITY ASSURANCE
APMC COLLEGE OF PHARMACEUTICAL EDUCATION &
RESEACH, HIMMATNAGAR

Pyrogen Testing
 Rabbit test (sham test)
 LAL Test (Bacterial Endotoxin Test)
Depyrogenation Techniques
 Physically removal of Pyrogens
 Inactivation of Pyrogens
Comparison of Pyrogen Test of IP, BP, USP.

A. Rabbit test (sham test)
B. LAL Test (Bacterial Endotoxin Test)

The test involves measurement of the
rise in body temperature of rabbits following
the intravenous injection of a sterile solution of
the substance being examined.
It is designed for products that can be tolerated
by the test rabbit in a dose not exceeding 10 ml
per kg injected intravenously within a period
of not more than 10 minutes.

Selection of animal
Materials:

Heated in oven at 250ºc for 30 min & 200ºc for
1 hour.
Diluents & Solutions Should be sterile &
Pyrogen free.

Recording of Temperature
Thermometer or thermistor.
Insert the thermometer or temperature sensing
probe into the rectum of the test rabbit to a
depth of about 5-cm. The depth of insertion is
constant for any one rabbit in any one test.

 PRELIMINARY TEST:
To the rabbit Inject intravenously 10 ml of
Pyrogen-free saline solution.

Record the temperatures of the animals,
beginning at least 90 minutes before injection
and continuing for 3 hours after injection of the
solution being examined.
Any animal showing a temperature variation of
0.6oC or more must not be used in the main
test.

 Preparation of the sample
 Procedure:
1. Carry out the test using a group of three rabbits.
2. Record the temperature of each animal. Not more than 40
minutes immediately preceding the injection of the test dose,
3. Record the "initial temperature" of each rabbit.( dT Sh’d not
>0.2)
4. Do not use any rabbit having a temperature higher than
39.8oC and lower than 38oC .
5. Inject the solution being examined slowly into the marginal
vein of the ear of each rabbit over a period not exceeding 4
minutes
9

6. The amount of sample to be injected varies according to
the preparation being examined and is prescribed in the
individual monograph.
7. The volume of injection is not less than 0.5 ml per kg and
not more than 10 ml per kg of body weight. Records the
temperature of each animal at half-hourly intervals for 3
hours after the injection.
8. The difference between the "initial temperature" and the
"maximum temperature" which is the highest temperature
recorded for a rabbit is taken to be its response. When this
difference is negative, the results is counted as a zero
response.

If the responses of the group of three rabbits in
which individual response is less than 0.6 oC , the
preparation being examined passes the test.
If the response of any rabbit is 0.6 oC or more, or if
the sum of the response of the three rabbits exceeds
1.4oC continue the test using five other rabbits.
If not more than three of the eight rabbits show
individual responses of 0.6oC or more, and if the sum
of responses of the group of eight rabbits does not
exceed 3.7oC , the preparation being examined passes
the test.

 It can be replicated and demonstrate the production of
fever in humans
 Detects all kinds of injectable Pyrogen unlike LAL test.

 DISADVANTAGE:
 Time consuming
 Expensive Procedure
 It is passfail test than assay
 It cannot be used to test certain drugs that depresses the
fever
 Tolerance to certain class of drugs can develop in rabbits
& also biological variations are observed

 LAL test = Limulus Amebocyte Lysate Test
 Test also referred by USP, IP, BP as BET test : Bacterial
Endotoxin Test

The addition of a solution containing endotoxins to a
solution of the lysate produces turbidity, precipitation or gel.
The rate of reaction depends on the concentration of
endotoxin , the pH and the temperature.
The reaction requires the presence of certain bivalent
cations , a proclotting enzyme system and clottable protein all
of which are provided by the lysate.

 Mechanism of Action:

Endotoxin 1-3-β- D-
Glucan

Factor C ---->
Factor G ---->
Factor C*
Factor G*

Factor B ---->
Factor B*

PRECLOTTING ENZYME ---->
CLOTTING ENZYME

COAGULOGEN ----> COAGULIN
15

The following six methods are described in the I.P. 2007 and
B.P. 2007
1) Method A: Gel clot method: Limit test
2) Method B: Gel clot method: Semi quantitative test.
3) Method C: Turbidimetric Kinetic method
4) Method D: Chromogenic kinetic method
5) Method E: Chromogenic end- point method
6) Method F: Turbidimetric end point method.
 the final decision is made based upon Method A , Unless
otherwise indicated in the monograph:

 Depyrogenate all glassware
 Time and temperature is required 30 minutes and 250 0C
 Plastic apparatus- which is free from detectable endotoxins

1) Water BET
2) 0.1M Hydrochloric acid BET
3) 0.1M Sodium hydroxide BET
4) Tris-chloride buffer pH 7.4 BET.
 Dissolve 0.6057 g of tris -(hydroxymethyl)
methylamine in 30 ml of water BET, add 0.33 ml of
hydrochloric acid, dilute to 100 ml with water BET and mix.
It gives a negative result under the conditions of the test.

5) Lysate:
A lysate of amebocytes from the horseshoe crab,
Limulus polyphemus, reconstituted as stated on the label.
For each batch, confirm the stated sensitivity as
prescribed under Sensitivity of the lysate.
6) Lysate solution
7) Reference Standard Endotoxin
8) The standard endotoxin stock solution
 Endotoxin is expressed in International Units (IU)
 1 IU=1 EU (ENDOTOXIN UNIT).

9) STANDARD ENDOTOXIN SOLUTIONS
10) PREPARATION OF THE TEST SOLUTIONS
Preparation of test solution by dissolving or diluting active
substance

Adjust the pH of test solution(about 6 to 8)

pH adjust with use of acid, base and buffer

Buffer must be validated to be free of detectable endotoxin

The Maximum Valid Dilution (MVD) is the
maximum allowable dilution of a sample at which the
endotoxin limit can be determined.
MVD = endotoxin limit × conc. of test solution
λ
λ = the labeled lysate sensitivity in the gel clot,
turbidimetric or chromogenic technique.

The Endotoxin limit= K/M
K = Threshold pyrogenic dose of endotoxin per kg of body
mass in single hour period.
M = Maximum dose of product per kg of
body mass in a single hour period
 The value of K is 5.0 EU/kg for injectable preparations
except for those administered intrathecally and is 0.2 EU/kg
for intrathecal preparations.

 The gel-clot technique allows detection or quantification of
endotoxins and is based on clotting of the lysate in the
presence of endotoxins.
 The concentration of endotoxins required to cause the lysate
to clot under standard conditions is the labelled lysate
sensitivity
 To ensure both the precision and validity of the test, confirm
the labelled lysate sensitivity and perform the test for
interfering factors as described under Preparatory testing

 SENSITIVITY OF THE LYSATE
Confirm in 4 replicates the labelled lysate sensitivity(λ)

Prepare standard solutions of at least 4 concentrations
equivalent to 2λ , λ , 0.5λ and 0.25λ from stock by diluting
with water for BET
Mix lysate solution with standard in each tube.(1:1)

Incubate the reaction mixture at 37 ± 1 °C for 60 ± 2 min
(avoiding vibration)
Invert it through approximately 180° in one smooth motion.

 If a firm gel has formed that remains in place upon
inversion, record the result as positive. A result is negative
if an intact gel is not formed.

The end-point is the last positive result in the series of
decreasing concentrations of endotoxin.

Calculate Geometric mean end-point concentration =
Where, ∑e = Sum of the log end-point concentrations.
f = number of replicates.
 The geometric mean end-point concentration is the
measured sensitivity of the lysate solution (IU/ml).
 If this is not less than 0.5λ and not more than 2λ, the
labelled sensitivity is confirmed

Solution Endotoxin Diluents Dilution Initial Number
conc./sol. to factor endotoxin of
which conc. replicate
endotoxin is
added
A None/Test - - - 4
(Sol. Of Prepa.) solution
B 2λ/Test Test 1 2λ 4
(Test for solution solution 2 1λ 4
Interference) 4 0.5λ 4
8 0.25λ 4
C 2λ/Water for Water for 1 2λ 2
(+ ve control) BET BET 2 1λ 2
4 0.5λ 2
8 0.25λ 2
D None/Water for - - - 2
(- ve control) BET 26

Prepare solutions A, B, C and D as shown in Table and use
the test solutions at a dilution less than the MVD, not
containing any detectable endotoxins, operating as described
under Confirmation of the labelled lysate sensitivity.
 The geometric mean end-point concentrations of solutions B
and C are determined.
The test is not valid unless all replicates of solutions A and D
show no reaction and the result of solution C confirms the
labelled lysate sensitivity

 If the sensitivity of the lysate determined with solution B is
not less than 0.5λ and not greater than 2λ, the test solution
does not contain interfering factors under the experimental
conditions used. Otherwise, the solution interferes with the
test
 If the preparation being examined interferes with the test at
a dilution less than the MVD, repeat the test for interfering
factors using a greater dilution, not exceeding the MVD
 Interference may be overcome by suitable treatment, such as
filtration, neutralization, dialysis or heat treatment.

Solution Endotoxin conc./solution to No. of
which endotoxin is added Replicates
A None/Test solution 2
B 2λ / Test solution 2
C 2 λ/water for BET 2
D None/Water for BET 2

 Prepare solutions A,B,C, and D as shown in table, and
perform the test on these solutions by following the
procedure described in Confirmation of the labeled lysate
sensitivity

 Prepare solution A and Solution B (Positive Product
control) using a dilution not greater than the MVD and
treatments Test for interfering Factors.

Interpretation:
 Ifa positive result is found for one of the test duplicates
and a negative result for the other, the test may be repeated
as described above. The results of the retest should be
interpreted as for the initial test.
 The substance or preparation being examined meets the
requirements of the test if the concentration of endotoxin is
less than the endotoxin limit stated in the monograph.

Solution Endotoxin Diluents Dilution Initial Number
conc. /solution factor endotoxin of
to which
conc. replicate
endotoxin is
added
A None/Test Water for 1 - 2
solution BET 2 - 2
4 - 2
8 - 2
B 2λ/Test solution - 1 2λ 2
(+ve control)
C 2λ/Water for Water for 1 2λ 2
BET BET 2 1λ 2
4 0.5λ 2
8 0.25λ 2
D None/Water for - - - 2

 The test is not valid unless the following three conditions
are met:
1. Both replicates of solution D(negative control) are
negative.
2. Both replicates of solution B(positive control) are positive
3. The geometric mean end-point concentration of solution C
is in the range of 0.5 λ to 2 λ.
 To determine the endotoxin concentration of solution A.
calculate the end-point concentration for each replicates
series of dilutions by multiplying each end-point dilution
factor by λ

 The endotoxin concentration in the test solution is the
geometric mean end point concentration of the replicates,
if the test is conducted with a diluted test solution
calculate the concentration of endotoxin in the original
solution by multiplying the results by dilution factor.
 If none of the dilution of the test solution is positive in a
valid test, record endotoxin concentration as less than λ .
 If all dilutions are positive, the endotoxin concentration is
recorded as equal to or greater than the greatest dilution
factor multiplied by λ

 The preparation meets the requirements of the test if the
endotoxin concentration is less than that specified in the
individual monograph.

1. Turbidimetric technique (Method C and F)
 This technique is a photometric test to measure the increase
in turbidity,
 Based on the test principle employed classified as,
 The end –point turnidimetric test (F)
 The kinetic- turbidimetric test (C)

2.CHROMOGENIC TECHNIQUES
(METHODS D &E)
 This techniques is used to measure the chromophore
released from a suitable chromogenic peptide by the reaction
of endotoxins with the lysate.
 Depending on the principle employed , this technique is
classified as
 End- point chromogenic test (E)
 The kinetic- chromogenic test.(D)

As the use of LAL test became more & more
prevalent, the FDA decided that single standardize
document was needed to govern all FDA regulated
products that were subject to LAL testing,
 An FDA task force was formed by representatives
from
1) Center for drug evolution & Research.
2) Center for biological evolution & Research.
3) Center for devices & Radiologic health.
4) Center for veterinary medicine.

Three basic requirements
i. The LAL reagent used in cell validation in
process and end product LAL tests must be
licensed by CBER
ii. The product manufacturer must perform an
initial Qualification of their LAL test laboratory
PERSONNEL
iii.Inhibition & enhancement tests must be
performed on test products do not interfere with
the enzymatic LAL endotoxin reaction.

A. Inhibition
 Occurs when the test recovers less endotoxin
Causes:
• Chemical nature of the drug product or excipients
• Factors that negatively effect serine protease enzyme
reaction such as High or low pH
• Oxidants or antioxidants
• Proteolytic agents
• High heat
• Chelating agents
• Inadequately dispersed purified endotoxin

B. Enhancement:
 Occurs if more Endotoxins recovered than expected
Causes:
 Chemical nature of the product
 Endotoxin contamination present in product
 Surfactants by increasing surface area of endotoxin.

 ADVANTAGE OF LAL TEST
 In vitro test ,
 More sensitive
 Easier to perform.
 Can give Quantitative results.
 Less time consuming.
 Less expensive.
 LIMITATION OF LAL TEST
 Specific for gram negative pyrogens only.
 Clotting enzyme is heat labile, pH sensitive.
 Possible interference Problems.

1) Pharmaceuticals:
 Parenteral dosage form
 Large volume Parenterals
 Small volume Parenterals

2) Biologicals
 In blood products & plasma fractions
 Vaccines

3)Medical Device
 Nebulizers used in Respiratory therapy

4)Diagnosis of disease caused by Gram –negative bacteria
5) In food & drinking water
6) Others: For validation of dry heat sterilization

INVITRO PYROGEN TEST (IPT):
 This test exploits the reaction monocytes/macrophages for
detection of pyrogens .
 Human whole blood taken from healthy volunteers is
incubated in presence of test sample, pyrogenic
contamination initiate the release of “the endogenous
pyrogen”
 Interlukin 1-β determined by ELISA after incubation.

(A)Removal of Pyrogens by physical Methods
(1) Dilution
(2) Ultra-filtration
(3) Reverse osmosis
(4) Distillation
(5) Adsorption on Charcoal
(6) Column Chromatography
(7) Charge Modified Media & Electrostatic Attraction
(8) Hydrophobic attraction to hydrophobic medium

(1) Dry heat sterilization
(2) Moist heat sterilization
(3) Use of dilute acids & Bases
(4) Oxidation
(5) Alkylation

Number of Maximum total peak Minimum total peak
Rabbits response (°C) to pass response (°C) to fail the
the test test
USP BP USP BP
3 1.4 1.15 1.4 2.65

6 - 2.80 - 4.30

8 3.7 - 3.7 -
9 - 4.45 - 5.95

12 - 6.60 - 6.60

 Encyclopedia of Pharmaceutical Technology,
Volume:13,By James Swarbick & James Boylan.
 Indian Pharmacopoeia 2007,Vol.-1,Appendice-1.
 British Pharmacopoeia 2007.
 United state pharmacopoeia.
 Remington( The science and practice of pharmacy),
Volume-I , Page no.562,832.

Pyrogen testing 112070804005

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