PYROGEN TEST

PRESENTED BY: PARTH KHANDHERIAM.PHARM SEM-2

DEPARTMENT OF PHARMACOLOGYL.M. COLLEGE OF PHARMACY

AHMEDABAD.

CONTENTS DEFINITION TYPES STRUCTURE SOURCES PYROGEN TEST

PYROGENS A Pyrogen is defined as “a fever producing agent” Metabolic products of Microorganisms. They are Soluble Filterable Thermostable Non Volatile Having nature

Endogenous (inside body)Exogenous (outside body)

Exogenous pyrogens They are foreign substance that are derived outside

the host. Lipopolysaccharides and other substances produce

d  by  pathogenic microorganisms. These substance becomes pyrogens when they are

administered parenterally to the host. They can be subdivided into the two group;

Microbial pyrogensNonmicrobial pyrogens

Endogenous Pyrogens Produced by the immune cells that are activated by

the presence of infectious agents (e.g. bacteria, viruses )

Endogenous pyrogens are usually cytokines, such as interleukin-6, interleukin-1, tumour necrosis factor, interferon-alpha, gp130 Receptor Ligands, and so on

How Endogenous Pyrogens cause FEVER

Bacterial endotoxin Pyrogen A toxin present inside a bacterial cell that is released

when it disintegrates. It is a high molecular weight complex which

constitute an integral components of the outer cell membrane of gram-negative bacteria.

It can exist in a cell-associated state or in a free state. Cell associated can be removed from a solution by

micro porous filters. But a free state easily passes through sterilizing

filters, heat stable.

Structure of endotoxin

The basic structure of endotoxin (LPS) is that of a Oligosaccharide covalently bound to a lipid component, called lipid A.

Oligosaccharide is composed of two parts;Core OligosaccharideO-antigen

The lipid A is the least variable components of Endotoxin, it consist of a disaccharide of glucosamine which is highly substituted with amide linked and ester linked long chain fatty acids.

Lipid A exerts its toxic effects when released from multiplying cells or when bacteria are lysed as a result of autolysis, ingestion and killing by phagocytes or certain types of antibiotics.

Sources of Pyrogen  Solvents, drugs, additives apparatus used in

manufacture, containers may be sources of pyrogens. The method of storage in between preparation and

sterilization also may cause the development of pyrogens.

Hence every item must be apyrogenic and method of storage must not allow any bacterial growth

Test for pyrogens

Rabbit test LAL TestIn Vitro

Pyrogen Test(IPT)

Leucocyte count

By measuring Electrical resistance

Pyrogen testing defines a process used by drug manufacturers to determine if bacterial toxins are present in vaccines and drugs that might cause fever when used on humans.

Rabbit test Qualitative fever response test

The test consists of measuring the rise in body temperature in healthy rabbits after the intravenous injection of a sterile test solution.

Why the Rabbit?

Other species not predictable Rabbit chosen for economic purposes Similar threshold pyrogenic response to humans Reproducible pyrogenic response

Requirements for the Rabbit Pyrogen Test

Rabbits must be healthy and mature New Zealand or Belgian Whites used mostly Either sex can be used Must be individually housed between 20 and 23°C Not varies more than ± 3º c. Free from disturbances likely to excite them. equipment and material used in test (glassware,

syringes, needles etc) must be free from Pyrogens by heating at 250º c for not less then 30 minutes or any other method

retaining boxes (comfortable for rabbits as possible)

Rabbits selected for testing on any given test day must not have been used for testing during two weeks.

General health status should be evaluted.

Preinjection Procedure

May not vary More than 3◦C.

To determine the correct dosage of test.

Procedure

Thermomistor probes or clinical thermometers are inserted rectally in the rabbits

If the probes remain throghout theTest period the rabbits must beRestrained with light fitting neck stocks.

A controlled temperature is determined not more than 30 min prior to injection of the test dose.

Volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight.

After injection rabbits temperature are recorded at 30 min intervals between 1 & 3 hour

If rabbit shows an individual temperature rise of 0.5◦C or more the test is continued with an additional 5 rabbits.

Interpretation of pyrogen test

<0.6◦CPreparation being examined passes the test.

>0.5◦C

Continue with

8

< Not >3.7◦C

Show individual

>0.6 ◦C

Preparation passes the test

Limitation of Rabbit test sensitivity variation For many drugs the volume tested is significantly

smaller. The rabbit test gives only a pass/fail result and is not

suitable for the control of endotoxin limit. It is not a quantitative test and less-well

standardized. A gap between the observed pyrogenicity in rabbits

and the expected pyrogenicity in humans. It is not suitable for all product categories. It is expensive. It involves the use of animals. Radiopharmaceuticals cannot be detected in rabbit

tests.

LAL Test

Limulus amebocyte lysate test. To measure the concentration of

endotoxins of gram-negative bacterial origin

Reagent: amoebocyte lysate from horseshoe crab, Limulus polyphemus

Principle

The addition of solution containing endotoxin to a solution of lysate produce turbidity.

The rate of reaction depends upon concentration of endotoxin , the pH and the temperature.

The endotoxin reference standard is the freeze dried.

The test is also based on the primitive blood-clotting mechanism of the horseshoe crab.

LAL Test• There are mainly Six methods used;

Method A. Gel-clot methodMethod B. Gel-clot method: semi-quantitative testMethod C. Turbidimetric kinetic methodMethod D. Chromogenic kinetic methodMethod E. Chromogenic end-point methodMethod F. Turbidimetric end-point method

Gel clot method• In presence of endotoxin a proenzyme in amoebocyte lyasate

is converted into an active form.

• The active enzyme then cleaves a clotting protein also found within the Limulus Amoebocyte.

Proenzyme Active EnzymeEndotoxin

Clotting Protein Clot Active

After incubation period reaction tubes are inverted.

If clot remains intact after inversion, the test is positive.

Turbidimetric technique This technique is a photometric test to measure the

increase in turbidity . Based on the test principle employed this technique

is classified as ;The end point-turbidimetric test (method-F)The kinetic-turbidimetric test (method-C)

• It is based on development of turbidity after cleavage of an endogenous substrate.

Method C :The kinetic-turbidimetric test

• The kinetic-turbidimetric test is a method to measure either the time needed for the reaction mixture to reach a predetermined absorbance or the rate of turbidity development.

Method F: The end point-turbidimetric

test

• The end-point turbidimetric test is based on the quantitative relationship between the endotoxin concentration and the turbidity of the reaction mixture at the end of an incubation period.

Chromogenic technique

• This is technique is used to measure the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the lysate.

• Depending on the test principle employed this technique is classified as;• The end-point chromogenic test (method E)• The kinetc chromogenic test (method D)

• It is based upon the development of colour after cleavage of a synthetic peptide chromogen complex.

• LAL reagent is formulated with a synthetic substrate gives yellow colour when acted upon by endotoxin activated enzyme.

• After cleavage of a synthetic peptide chromogen

Method E: The end-point chromogenic

test

• The end-point chromogenic test is based on the quantitative relationship between the endotoxin concentration and the quantity of chromophore released at the end of an incubation period.

Method D: The kinetic chromogenic

test

• The kinetic chromogenic test measure either the time needed for the reaction mixture to reach a predetermined absorbance or the rate of colour development.

Applications of LAL test1.Pharmaceuticals In parenteral dosage form Large volume parenteral Small volume parentral

2. Biological In blood product and plasma fraction Vaccine Injectables, biological products, surgical devices or

medical devices and renal dialysis fluids can be tested for absence of pyrogens by LAL test.

(1,3)-β-D-glucan, a material found in fungal cell walls, plant tissue can be detected by LAL test.

3. Diagnosis Disease caused by gram (-)ve bacteria urinary tract infections and spinal meningitis.

4. Medical device• British pharmacopoeia(2007)-Appendix XIV-C-A-331• Indian pharmacopoeia(1996)-VOL:II-A-24• Encyclopedia of PHARMACEUTICAL TECNOLOGY:

edited by James swarbrick –(2007)- vol -5 pg no:3056.

• nebulizer used in respiratory therapy

5.The LAL test has also been used to assess food spoilage, air and water quality.

6. For validation of dry heat sterilization

7.LAL test is also assay of choice for researchers studying both the clinical and environmental effects of endotoxin.

ADVANTAGES OF LAL TEST

Less variable In vitro test Easier to perform More sensitive Less Expensive Less Time consuming Can give Quantitative result

LIMITATION OF LAL TEST

Specific for gram (-)ve pyrogen only Clotting enzyme is heat labile, pH sensitive Possible interference problem

INVITRO PYROGEN TEST (IPT)Test Technology

• The IPT is an ELISA assay that utilizes fresh or cryopreserved human whole blood.

• Immune cells wlthin the blood will recognize exogenous pyrogen and in response release fever-inducing signal molecules that can then be measured.

Alternative of Rabbit Pyrogen Test

Limitatians of the rabbit pyrogen test include Sensitivity variations It is also not possible to test certain drugs such as

cytokines, antibiotics, certain sedatives/analgesics, plasma proteins and radiopharma- ceuticals.

The rabbit pyrogen test does not always identify human pyrogens.

IPT ASSAY STEPS1.Prepare BloodDraw fresh blood in heparinized tubes. Blood must be used within four (4) hours of collection.

2. Prepare ReagentsPrepare Standards, Positive & negative controls and samples following package inserts and Certificates of Analysis.

3. Toad samples, Standards and ControlsUsing the tissue culture microplate, add 0.2 mL NaClto wells f‹›r standards, negative controland 0.18 mL NaCI to wells for positive sample controls. Next add 0.02 mL of samples and controls to designated wells.

4. Load Blood and Incubate SampleAdd 0.02 ml of blood to each well. Mix tl›e welJs thoroughly using a plate mixer and incubate overnight at 37°C. The next day, remove from incubator and mix the plate until cells are resuspended. Allow cells to settle for approximately 45 minutes.

5. Transfer to Coated plateUsing the antibody-coated microplate, add 0.1 mL of conjugate to each well. Add 0.1 mL of supernatant to antibody plate. Incubate the plate on a plate mixer at room temperature for 90 minutes.

6. Add SubstrateWash the plate 4-5 times with wash solution until clear, tapping plate between washes to remove excess wash solution. Add 0.2 mL of TMB substrate to each well.Incubate at room temperature in the dark for 30 minutes. Add 0.1 mL of STOP solution to all wells. Read plate at 450 nm within 15 minutes.

Samples are considered pyrogenic if OD readings of Sample are

> OD reading of 0.5 EU/mL Standard.

By leucocyte count Injection of pyrogens causes changes in the white

cells picture. IF Pyrogens present. Fall in small lymphocytes Rise in young neutruphills.

By measuring electrical resistance

Pure water has very high resistance. If Pyrogens are present they will carry minerals and

decrease in resistance occurs . Resistance measured by conductivity meter.

REFERENCES Indian pharmacopoeia 2007, Government of India:

Ministry of health & Family welfare, Vol-1, page no:34 Williams Kevin L., Endotoxins Pyrogens, LAL testing

and Depyrogenation, Volume 167, 3rd edition. Sushruta Mulay et al, AN OVERVIEW OF LIMULUS

AMOEBOCYTE LYSATE (LAL) TEST, International Research Journal Of Pharmacy 2011; 67-71.

U.S.PHARMACOPEIA Pharmaceutical Formulations by M.E.Aulton, H.C.

Ansel.page no 195-196.