Pyrogen and its various test

PREPARED AND PRESENTED BYBARBHAIYA PRACHI M., ([email protected])

DEPARTMENT OF QUALITY ASSURANCE (M.Pharm-1)

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A-ONE PHARMACY college, ENASAN

ENDOTOXIN TEST ING COMPARISON AS PER IP,BP & USP

Research Articles Based Presentation on

Objectives of the studyO Pharmaceutical products which are injected into the human body

are tested for pyrogenic substances. The most common, and arguably most important test, is for bacterial endotoxin. The pathological effects of endotoxin, when injected, are a rapid increase in core body temperature followed by extremely rapid and severe shock, often followed by death before the cause is even diagnosed. The purpose of the LAL testis to detect endotoxin. The goal of a BET validation is to find a valid, compatible range (concentration or dilution) for routine LAL testing of the material in question.

One get become informed about:O Regulatory requirementsO The different testing methodsO Use of the methods in daily routineO Solutions for issues with interferences, inhibitors and maskingO Testing of packaging materialsO Application of alternative testing methods

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Summary Report of Research Article Based Learning

LIST OF 5 RESEARCH ARTICLE/REVIEW ARTICLE SELECTED FOR THIS STUDY

1) Vipond C, Findlay L, Feavers I. “Limitations of the Rabbit Pyrogen Test for Assessing Meningococcal OMV based Vaccines”. ALTEX. 2015: 1-9. http://www.altex.ch/Online-first.95.html

2) Aakanksha B, Monika B, Anil B. “An overview on pyrogen reduction methods”. Int R. J Pharm.. 2010, 1(1): 57-61.

3) Sushruta M, Kale A. “An overview of Limulus Amoebocyte Lysate Test”. Int R. Jour of Pharmacy. 2011, 2(4): 67-71.

4) Horiuchi Y, Yuen CT, Ochiai M. “Strategic Approaches for Developing Alternative Tests for Safety and Potency of Vaccines”. Procedia in Vaccinology. 2011, 5(2): 156-163.

5) Rockel C, Hartung T. “Systematic Review of Membrane Components of Gram-Positive Bacteria Responsible as Pyrogens for Inducing Human Monocyte/Macrophage Cytokine Release”. Frontiers in Pharmacol. 2012, Vol 3, Article 56: 1-19.

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1. Limitations of the rabbit pyrogen test for assessing meningococcal OMV based

vaccinesO The test is a current Pharmacopeial method

and is used to evaluate the pyrogen content of some vaccines. The method is unsuitable as a safety test for products due to the high levels of endotoxin present in the vaccine which generate a pyrogenic response in rabbits when delivered intravenously without any dilution. Its use as a consistency test is also ambiguous as the test gives a qualitative rather than quantitative response and the rabbit models are highly variable. In addition there is evidence that measuring the temperature rise of the animals over three hours does not capture the maximum fever response.


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2. An overview on pyrogen reduction methods

O Rabbit testThis test is official in Indian Pharmacopoeia, British Pharmacopoeia and United State Pharmacopoeia. The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination. 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.O Limulus amebocyte lysate test (LAL test)This test is official in United State Pharmacopoeia. It is 5-10 times more sensitive than the rabbit test. It is vitro test method for pyrogens has been developed utilizing the gelling property of the lysate of the amebocyte of limulus polyphemus. In the presence of pyrogeneic endotoxins from gram negative bacteria, a firm gel is formed within 60 minutes when incubated at 37°C.


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3. An overview of Limulus Amoebocyte Lysate Test

O KINETIC AND ENDPOINT TURBIDIMETRIC ASSAY During the LAL-endotoxin reaction, the solution mixture becomes increasingly turbid. The kinetic turbidimetric assay (KTA) requires an incubating microplate or tube reader driven by endotoxin specific software. The reaction mixtures in a KTA system are continually monitored for changes in optical density in each sample that is caused by scattering and absorption of light. Generally, the KTA method measures the onset time needed to reach a predetermined absorbance by each reaction mixture.O KINETIC AND ENDPOINT CHROMOGENIC ASSAYS Endotoxin activates enzymatic cascade from the reagent, the clotting enzyme reacts with a synthetic substrate that has been added to the reaction mixture. The substrate consists of colorless amino acid chain attached to a chromophore. The activated clotting enzyme cleaves the bond that holds the chromophore to amino acid. The amount released is proportional to the concentration of amino acid. The chromophore that is released changes the color of reaction mixture, thereby increasing the optical density.


4. Strategic approaches for developing alternative tests for safety and potency of vaccines

O Safety control tests for vaccines need to be capable of quantifying in vivo toxicity of vaccines. Endotoxin contamination is a threat for vaccine safety and has traditionally been controlled using either an in vivo rabbit pyrogen test or the in vitro Limulus amebocyte lysate (LAL) assay. An in vitro pyrogen test was developed using prostaglandin E2 induction in rabbit peripheral blood. Responsiveness of LAL and rabbits to various endotoxins, however, differ from that of humans. A monocyte activation test was developed using human monocytes but is not applicable where the use of donated blood is limited to therapeutic purposes. Therefore, a clinically relevant in vitro pyrogen test was developed using a human monocytic cell line responsive to various pyrogens in a manner consistent with that of human peripheral blood.

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5. Pyrogens for inducing Human monocyte/macrophage cytokine release


O Fifty years after the elucidation of lipopolysaccharides (LPS, endotoxin) as the principal structure of Gram-negative bacteria activating the human immune system, its Gram-positive counterpart is still under debate. Pyrogen tests based on the human monocyte activation have been validated for LPS detection as an alternative to the rabbit test and, increasingly, the limulus amebocyte lysate test. For full replacement, international validations with non-endotoxin pyrogens are in preparation. Following evidence-based medicine approaches, a systematic review of existing evidence as to the structural nature of the Gram-positive pyrogen was undertaken.

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SUMMARY OF 5 ARTICLESBasically there are test performed to detect the presence of pyrogens in sterile parenteral products they are 1) Rabbit Test 2) LAL Test.O Rabbit test:- This test basically involves the injection Sample

solution which is to be tested into a Rabbits Which are use as test animals through ear vein. The Temperature sensing probe (Clinical Thermometer, Thermistor or similar probe) into a rectum cavity of Rabbit at the depth of 7.5 cm, the test solution must be warmed at 37 degrees prior to injection. Then Rectal temperature is recorded at 1,2,3 hr. subsequent to injection. This test is performed in separate area designed solely for this purpose under environmental conditions similar to animal house should be free from disturbances that likely to excite them. Initially this test is performed on 3 Rabbits but if required results are not obtained this test is repeated on 5 additional Rabbits with same sample solution administer to initial 3 rabbits. Prior to 1hr of injecting sample solutions the control temperatures of rabbits are determined. Use only those rabbits whose control temperature is no vary by more than 1 degree Celsius.


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*Interpretation:- The solution is judged to be non pyrogenic if no single rabbit show rise in temperature of 0.5 degree Celsius but if this condition is not met then the test if repeated on 5 additional rabbits with same preparation administer.

O LAL test:- It is an recently developed in vitro test method for pyrogen utilizing gelling property of lysates of amebocytes of limulus polyphemus which is found only at specific locations along the east coast of North America and along southeast Asia. It is derived from horse shoe crab; the basic procedure is the combination of 0.1 ml of test sample with LAL Reagent after incubation for 1 hr. at 37 degree Celsius the mixture is analysed for the presence of Gel clot. The LAL Test is positive indicating that the presence of endotoxin. Its applications are mainly to Pharmaceutics, Biological, devices, disease states, food, and validation of heat cycles. This method has several advantages of Rabbit test they are Greater sensitivity and reliability specificity, less variation, wider application, less expensive and simplicity. Further The following 5 methods are described

O Method A. Gel-Clot Limit Test Method O Method B. Semi-quantitative Gel-Clot Method O Method C. Kinetic Turbidimetric Method O Method D. Kinetic Chromogenic Method O Method E. End-Point Chromogenic Method


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CONCLUSIONO Quality control should be a fundamental segment of

parenteral products manufacturing. All of the 4 basic tests which are performed are essential and have its own importance in parenteral production. All of these tests ensure that product meet its quality which has been judged to satisfactory also. Each test is unique and provides detailed assessment of quality control for parenteral products.

O Although rabbit test exist for the detection of endotoxin, the LAL test is most effective because it is capable of detecting endotoxins qualitatively as well as quantitatively (As little as one millionth of a billionth of a gram of endotoxin. LAL test is currently recognized by several major pharmacopoeias and is used worldwide. LAL test has proven its usefulness in not only to detect harmful effects of endotoxins (as pyrogens) in pharmaceutical products but has become an indispensable tool in controlling endotoxin in process and equipment used to produce pharmaceuticals.


Pharmaceutical aspectsComparison of Pyrogen test of IP, BP, USP Interpretation of dataComparison of LAL and other pyrogen

testsReferences

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POINTS TO BE COVERED:

PHARMACEUTICAL ASPECTSO Pyrogens Definition:

Pyrogens are fever producing substances, which are metabolic products of micro-organisms.1

Chemically, they are lipid substances associated with a carrier molecule, which is usually a polysaccharide. The carrier may also be a peptide. These carriers increase the solubility of the lipid.1

Pyrogens are produced by many micro-organisms including bacteria, yeasts and moulds. Most potent pyrogens are the endotoxins produced from the cell walls of the gram-negative bacteria. (eg. Pseudomona sp., salmonella sp., E.coli)

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Cont…O Sources of Pyrogens2:

Apparatus and containers used in preparation

SolventsDrugsAdditives

OClassification of Pyrogens2:1. Exogenous Pyrogen (Exotoxins)2. Endogenous Pyrogen (Endotoxins)

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Cont… The bacterial substance lipopolysaccharide (LPS),

present in the cell wall of some bacteria, is an example of an exogenous pyrogen.

Pyrogenicity can vary: In extreme examples, some bacterial pyrogens known as superantigens can cause rapid and dangerous fevers.

O Depyrogenation may be achieved through filtration, distillation, chromatography or inactivation.3

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DIFF. BETWEEN PYROGEN & ENDOTOXIN

PYROGEN= PYRO + GEN ENDOTOXIN = ENDO + TOXIN

O A pyrogen is a substance specifically inducing fever.

O An endotoxin is a toxin released in the presence of some gram-negative bacteria e.g. salmonella.

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PYROGEN TEST:4 OFFICIAL TEST According to IP RABBIT TEST (IN VIVO)

OTHER TESTS 1.) LAL TEST (IN VITRO) 2.) HUMAN WHOLE BLOODTEST (WBT) 3.) LEUCOCYTE COUNT ADVANCED TECHNIQUE ENDOLISA

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Rabbit Test (in vivo test)

The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination. 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. 19

The rabbit as an animal model for pyrogen testing:

1) Inexpensive. 2) Easy to handle. 3) Similar Physiological Characteristics.4) Elevation of body temperature is

detectable.

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Test animalsUse healthy, adult rabbits of either sex,

preferably of the same variety, weighing not less than 1.5 kg.

fed on a complete and balanced diet and not showing loss of body weight during the week preceding the test.

House the animals individually in an area of uniform temperature (± 2º), preferably with uniform humidity, and free from disturbances likely to excite them.

Do not used the same animal for new test before 48 hrs.

If used animal shows a temp.greater than 0.6 c or the substance under examination was found to be pyrogenic, at least 2 weeks must be allowed to elapse before the animals is used again.

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MaterialsAll glassware, syringe and needles must be

thoroughly washed with water for injection and heated in a hot air oven at 250℃ for 30 min. or at 200℃ for 1 hour.

The retaining boxes for rabbits in which the temperature is being measured by electrical device should be made in such a way that the animals are retained only by loosely-fitting neckstocks and the rest of the body remains relatively free so that the rabbits may sit in a normal position.

The animals must be put in the boxes 1 hour before the test and remain in them throughout the test.

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Recording of temprature Use an accurate temperature-sensing device

such as a clinical thermometer or thermistor or other suitable probes that have been calibrated to assure an accuracy of 0.1º and have been tested to determine that a maximum reading is reached in less than 5 minutes.

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.

If an electrical device is used, it should be inserted in the rectum of the rabbit 90 minutes before the injection of the solution being examined and left in position throughout the test.

After a period of time not less than that previously determined as sufficient, record the rabbit’s body temperature.

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A) PRELIMINARY TEST (SHAM TEST)

1.) If animals are used for the first time in a pyrogen test or have not been used during the 2 previous weeks, condition them 1 to 3 days before testing the substance under examination by injecting intravenously into them 10 ml per kg of body weight of a pyrogen-free saline solution warmed to about 38.5º.

2.) 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.

3.) Any animal showing temperature variation of 0.6º or more must not be used in the main test.

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B) MAIN TEST Carry out the test using a group of three

rabbits.Preparation of the sample: Dissolve the substance under examination in,

or dilute with, pyrogen-free saline solution or other solution prescribed in the monograph.

Warm the liquid under examination to approximately 38.5º before injection.

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Procedure1.) Record the temperature of each animal.2.) Record the “initial temperature” of each

rabbit, which is the mean of two temperatures recorded for that rabbit at an interval of 30 minutes.

3.) Inject the solution under examination slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph.

4.) Record the temperature of each animal at half-hourly intervals for 3 hours after the injection.

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Cont…5.) 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.

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INTERPRETATION OF DATA If no rabbits shows individual temp. rise

of 0.6 c or more ͦ or sum of 3 individual max. temp NMT 1.4 c ͦ No pyrogen (passes the test).

If both occurs > Repeat the test with 5 more rabbits.

Out of 8 rabbits if NMT 3 shows individual rises in temp. of 0.6 c or more ͦ & if sum of 8 individual max. temp. do not exceed 3.7 c ͦ No pyrogen (passes the test).

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Advantages: The human and rabbits are equally responsive

to threshold levels of the pyrogens It is used to identify the presence of a wide

range of pyrogens.Disadvantages: Time consuming. Expensive procedure. Require skilled animal handler and technicians. Can not be used to test certain drugs that

depresses the fever.

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COMPARISON OF PYROGEN TEST OF BP, IP & USP6,7,8

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LIMULUS AMOEBOCYTE LYSATE TEST (LAL TEST)/

BACTERIAL ENDOTOXIN TEST (BET)/

IN VITRO PYROGEN TEST O AIM: LAL test is to measure quantitatively the

amount of bacterial endotoxins in a given sample of parenteral.

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Principle:

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Preclotting enzyme

(Present in lysate)

ENDOTOXIN

ACTIVATED CLOTTING ENZYME

Soluble Coagulogen(Clottable

protein present in

lysate)

Insoluble coagulin(Gel clot)

Gel

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The following 5 methods are described

O Method A. Gel-Clot Limit Test Method O Method B. Semi-quantitative Gel-Clot

Method O Method C. Kinetic Turbidimetric Method O Method D. Kinetic Chromogenic Method O Method E. End-Point Chromogenic

Method

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THREE TECHNIQUES4

Gel Clot technique

• Which is based on formation of Gel

Turbidimetric technique

• Which is based on turbidity

Chromogenic technique

• Which is based on develop-ment of color

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Determination of maximum valid dilution

MVD-Maximum allowable dilution of sample at which endotoxin limit can be determine by following formula,

MVD = Endotoxin limit × Conc. of the testsolution

λ where, λ is the labelled sensitivity of the

lysate (EU/ml).Concentration of the test solution is

expressed as Mg/ml in case the endotoxin limit is specified by weight (EU/mg), or as Units/ml in case the endotoxin limit is specified by Unit (EU/Unit), or as 1.0 ml/ml in case the endotoxin limit is specified by volume (EU/ml).

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Gel clot methodMethods A and B depend on the formation of a firm

gel when a solution containing bacterial endotoxins is incubated after mixing with the lysate.

Method A is conducted as a limit test wherein both the replicate solutions of the preparation under examination must contain endotoxin in the concentration less than the endotoxin limit concentration specified in the individual monograph.

Method B determines the endotoxin concentration semiquantitatively in the preparation under examination.

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METHOD A: GEL CLOT LIMIT TEST

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Preparation of test solution Prepare the solutions and dilutions with

water BET. adjust the pH of the solution under

examination to 6.0 to 8.0 using sterile 0.1M hydrochloric acid BET, 0.1M sodium hydroxide BET or a suitable buffer prepared with water BET.

Prepare the sample solution at any dilution at or below MVD.

Use water BET as negative control (NC) and two positive controls.

One of the positive controls consists of the CSE at a concentration of 2λ and the other consists of the test solution spiked with CSE to give a concentration of 2λ.

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Interpretation of result If the positive product control is positive

and the negative control as well as the test solutions are negative complies with the bacterial endotoxin test.

The test is not valid if the positive product control is negative or if the negative control is positive.

Meets the requirements of the test if the endotoxin content is less than the endotoxin limit stated in the individual monograph. 41

Retests If a positive result is found for one of the test

solution 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.

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METHOD B: SEMI QUANTITATIVE GEL CLOT METHOD

Preparation of test solutions. Prepare test solutions at concentrations

of MVD, 0.5 MVD, 0.25 MVD or any other appropriate dilutions relative to the dilution at which the test for interfering factors was completed.

Additionally, prepare a similar series of test solutions spiked with 2λ of CSE each (PPC).

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CALCULATION AND INTERPRETATION OF RESULT

Calculate the endotoxin concentration in the product, determine for the series of test solutions the lowest concentration or the highest dilution giving a positive (+) reaction.

Multiply this dilution factor with λ to obtain the endotoxin concentration of the product.

For instance, if MVD is equal to 8 and the positive reaction was obtained at 0.25 MVD and l was equal to 0.125 EU/ml,

The endotoxin concentration in the test solution will be 8 × 0.25 ×0.125 = 0.25 EU/ml.

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Cont… If none of the dilutions of the series gives a positive

reaction, the endotoxin concentration will be less than the value obtained by multiplying the lowest dilution factor with λ.

If all the dilutions of the series give a positive reaction, the endotoxin concentration will be more than the value obtained by multiplying the highest dilution factor with λ.

Calculate the endotoxin content of the product under examination from the endotoxin concentration.

The product under examination meets the requirements of the test if the endotoxin content is less than the endotoxin limit stated in the individual monograph.

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Quantitative Method1) Kinetic turbidimetric method (Method C),2) Kinetic chromogenic method (Method D)

and3) End-point chromogenic method (Method

E).

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Photometric method

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Kinetic turbidimetric

method(method c)

Kinetic chromogenic

method(method D)

Measuring either the time (onset time) needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development.

Measuring either the time (onset time) needed to reach a predetermined absorbance of the reaction mixture or the rate of colour development.

End point chromogeni

c method (method e)

Measuring the colour intensity at the end of an incubation period after the reaction is stopped by the addition of a suitable acid.

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Standard Curve End point Test

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LAL Assay5

Gel-Clot Colorimetric

Turbidimetric

Semi-quantitative

Quantitative Quantitative

Simple, Least expensive

Requires incubating

plate or tube reader

Requires incubating

plate or tube reader

Manually read and recorded

Can be automated

Can be automated

Sensitive down to 0.03

EU/ml

Sensitive down to

0.005 EU/ml

Sensitive down to

0.001 EU/ml

ADVANTAGES OF LAL TEST1) It is in-vitro and does not require animal handling, thus

is more convenient2) It is 10 times more sensitive than that of the in-vivo

rabbit test3) Less expensive.4) Less time consuming, i.e., 1 vs 3 hours required by

rabbits test5) It requires less laboratory facilities and minimum

equipments6) It requires less test volume7) Less variable.8) Easier to perform9) Can give quantitative result.

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ADVANTAGE OF LAL TEST OVER RABBIT TEST

1) LAL Test is used to quantify the endotoxin count where as rabbit test is done to detect the presence of pyrogens.

2) Less time consuming(within 1 hr.)3) Simple and economic.

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LIMITATION OF LAL TEST1) Specific for gram (-)ve pyrogen only2) Clotting enzyme is heat labile, pH

sensitive3) Possible interference problem

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APPLICATION5

1) Pharmaceuticals In parenteral dosage form2) Biological In blood product and plasma fraction Vaccine3) Medical device nebulizer used in respiratory

therapy.4) Diagnosis of disease caused by gm-ve

bacteria.5) In food and drinking water.

COMPARISON OF THREE PYROGEN TESTS9:

TEST RABBIT LAL WBTPyrogens Bacteria gram

negative+ + +

Bacteria gram positive

+ - +

fungi + - +Application Biologicals + - +

Pharmaceuticals + + +

Medical devices - + +

Air quality - (+) +Blood components

- - +

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REFERENCES1) Vipond C, Findlay L, Feavers I. “Limitations of the Rabbit

Pyrogen Test for Assessing Meningococcal OMV based Vaccines”. ALTEX. 2015: 1-9. http://www.altex.ch/Online-first.95.html

2) Aakanksha B, Monika B, Anil B. “An overview on pyrogen reduction methods”. Int R. J Pharm.. 2010, 1(1): 57-61.

3) Sushruta M, Kale A. “An overview of Limulus Amoebocyte Lysate Test”. Int R. Jour of Pharmacy. 2011, 2(4): 67-71.

4) Horiuchi Y, Yuen CT, Ochiai M. “Strategic Approaches for Developing Alternative Tests for Safety and Potency of Vaccines”. Procedia in Vaccinology. 2011, 5(2): 156-163.

5) Rockel C, Hartung T. “Systematic Review of Membrane Components of Gram-Positive Bacteria Responsible as Pyrogens for Inducing Human Monocyte/Macrophage Cytokine Release”. Frontiers in Pharmacol. 2012, Vol 3, Article 56: 1-19.

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Cont…6) “Indian Pharmacopoeia 2007”,Government of India, Ministry Of Health and Family Welfare, Controller Of Publication, New Delhi Volume-1,Appendex-2.2.9, Page No.34-35.7) British Pharmacopoeia 2007.8) United State Pharmacopoeia 2007.

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Thank you

Special THANKS to

Ms. Bhumika Sakhreliya

Pyrogen and its various test

Healthcare