QUALITY CONTROL OF PHARMACEUTICAL DOSAGE FORMS:-

PYROGEN TESTING

Presented by:

Md. Mehedi Hasan

Lecturer, Department of Pharmacy, UITS

“Parenterals are the sterile dosage form intended for administration other than the

enteral route and exert their action by directly entering into the systemic circulation”

Pharmaceutical Microbiology

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Pyrogens

Pyrogens are the by-products of microorganisms mainly of

bacteria, molds and viruses.

During the processing these pyrogens may come from water,

active constituent or the excipient or from the equipments.

Chemically these pyrogens are lipid substances associated with

carrier usually polysaccharides or may be proteins.

Having nature

Endogenous (inside body)

Exogenous (outside body)

Exogenous pyrogens –

mainly lipopolysaccharides

bacterial origin, but not necessary

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Endotoxin characteristic

thermostable

water-soluble

unaffected by the common bactericides

non-volatile

These are the reasons why pyrogens are difficult to

destroy once produced in a product

Unit of Endotoxin: Endotoxin is expressed in

International Unit

1 IU = 1EU (Endotoxin Unit)

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Test for pyrogens = Rabbit test

the development of the test for pyrogens reach

in 1920

a pyrogen test was introduced into the USP XII

(1942)

The test consists of measuring the rise in body

temperature in healthy rabbits by the

intravenous injection of a sterile solution of the

substance under the test.5

Why the Rabbit?

Reproducible pyrogenic response

Other species not predictable

Similar threshold pyrogenic response to

humans

Rabbit chosen for economic purposes

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Rabbit Pyrogen Test

Rabbits must be healthy and mature

New Zealand or Belgian Whites used mostly

Either sex may be used (male or female)

Must be individually housed between 20 and 23°C

Not varies more than ± 3º c.

Free from disturbances likely to excite them.

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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)

Thermometers or thermistor probe (standardized

position in rectum, precision of ± 0.1°C)

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Rabbit pyrogen test

Preliminary test (Sham Test)

intravenous injection of sterile pyrogen-free

saline solution

Warm the pyrogen free solution up to 38.5ºc

to exclude any animal showing an unusual

response to the trauma (shock) of injection

any animal showing a temperature variation

greater than 0.6C is not used in the main test

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Rabbit pyrogen test -

main test:

group of 3 rabbits

preparation and injection of the product:

warming the product

dissolving or dilution

duration of injection: not more than 4 min

the injected volume: not less than 0.5 ml per 1 kg and not more than 10 ml per kg of body mass

determination of the initial and maximum temperature

all rabbits should have initial Temperature: from 38.0 to 39.8C

the differences in initial Temperature should not differ from one another by more than 1C

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Interpretation of the results:

the test is carried out on the first group of 3

rabbits; if necessary on further groups of 3

rabbits to a total of 4 groups, depending on the

results obtained

intervals of passing or failing of products are

on the basis of summed temperature response

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The result of pyrogen test:

No.of Rabbits Individual Tempt. rise

(°c)

Tempt. Rise in group (°c)

Test

3 rabbits 0.6 1.4 Passes

If above not passes

3+5 = 8 rabbits

0.6 3.7 Passes

If above test not passes the sample is said to pyrogenic.

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Comparison of pyrogen test of BP & USP

Number of rabbits

Maximum total peak response (0 C) to pass the test

Minimum total peak response (0 C) to fail the 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 - 6.6014

LAL Test

Limulus amebocyte lysate test.

to measure the concentration of endotoxins of

gram-negative bacterial origin

reagent: amebocyte lysate from horseshoe crab,

Limulus polyphemus

Limulus-Amebocyte Lysate is prepared by bleeding

healthy mature specimens by heart puncture.

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Application of LAL test1) Pharmaceuticals:

- Parenteral dosage form

- Large Volume Parenterals (LVP)

- Small Volume Parenterals (SVP)

2) Biologicals:

- In blood products & plasma fraction

- Vaccines

3) Medical devices:

- Nebulizer and Respiratory therapy

4) Diagnosis of disease caused by Gram-negative bacteria

5) In food and Drinking water

6) Other: for validation of dry heat sterilization

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Advantages of LAL test

1. It is in-vitro and does not require animal handling, thus is more convenient

2. It is 10 times more sensitive than that of the in-vivo rabbit test

3. It is economical

4. It consume less time, i.e., 1 vs 3 hours required by rabbits test

5. It requires less laboratory facilities and minimum equipments

6. It requires less test volume

7. It is more accurate

Disadvantages:

1. This test detects Gram negative pyrogens only

2. Clotting enzyme is heat labile, PH sensitive

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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 based on the primitive blood-

clotting mechanism of the horseshoe crab20

Commercially derived LAL reagents

bleeding adult crabs into an anticlotting solution

washing and centrifuging to collect the amebocyte

lysing in 3% NaCl

lysate is washed and lyophilized for storage

activity varies on a seasonal basis and standardization is necessary.

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Process flow of LAL Test

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Horseshoe crab are donating their blue blood to create LAL for endotoxin testing

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Test performance (short)

avoid endotoxin contamination

Before the test:

interfering factors should not be present

equipment should be depyrogenated

the sensitivity of the lysate should be known

Test:

equal volume of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-tube

incubation at 37°C, 1 hour

remove the tube - invert in one smooth motion (180°) - read (observe) the result

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Endotoxin concentration monitoring• Following method are used to monitor the endotoxin

concentration

– Method A: gel-clot method: limit test

– Method B: semi-quantitative gel-clot method

– Method C: kinetic turbidimetric method

– Method D: kinetic chromogenic method

– Method E: end-point chromomeric method

different techniques:

the gel-clot technique - gel formation

the turbidimetric technique - the development of turbidityafter cleavage of an endogenous substrate

the chromogenic technique - the development of colorafter cleavage of a synthetic peptide-chromogen complex.25

Other pyrogen tests

In-vitro pyrogen tests (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, pyrogeniccontamination initiate the release of “the endogenous pyrogen”

Interlukin 1-β determined by ELISA after incubation.

Enzyme-Linked Immunosorbent Assay, an immunologicalassay technique making use of an enzyme bonded to aparticular antibody or antigen.

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De-pyrogenation(A) Removal of pyrogen by physical methods1. Dilution2. Ultra-filtration (WFI preparation) (Fiber Polysulphone membrane)

3. Reverse osmosis (WFI preparation)4. Distillation (WFI preparation)5. Adsorption on charcoal6. Column Chromatography7. Charge modified media and electrostatic attraction8. Hydrophobic attraction to hydrophobic medium

(B) Inactivation of pyrogen1. Dry heat sterilization (glassware 2200C)2. Moist heat sterilization3. Use of dilute acid & base4. Oxidation5. Alkylation 27

Endotoxin Limit Calculation (ELC)ELC =

K = maximum acceptable bacterial endotoxin content/kg bodymass (this is a constant; for injectables, it is 5 EU/kg)

M = maximum daily dose of the tested products/kg body mass(calculated with reference to the body mass of 70 kg)

Example, For Diclofenac Sodium injection:K = 5 EU/Kg; M = 2.143 mg/Kg

So, ELC =

Answer:

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Common References from BP

• The British Pharmacopoeia (2002) has a limit:

0.25 IU/ml in Glucose Intravenous Infusion;

• this value is similar for many BP intravenous infusions.

• As another example insulin should contain

10IU/mg of endotoxin.

The endotoxin limit for drugs gaining access to the CSF is reduced to 0.2 EU/kg because the intrathecal (IT) route is the most toxic route for endotoxins.

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Freeze-drying/Lyophilization• Freeze-drying is an aseptic process whereby water is removed

from a frozen product mainly by sublimation, i.e. by theconversion of ice directly into the vapour state without theintermediary of liquid water. It is a batch process, of relativelylong duration, and is used frequently for drugs of poor stability.Drugs are reconstituted into solution immediately prior toinjection.

The process consists of three stages:

• Freezing, which slows down degradation and solidifies the product.

• Primary drying whereby energy is provided to the system and a vacuumapplied to expedite the removal of moisture at sub-ambienttemperatures.

• Secondary drying, whereby the product is heated to remove the lasttraces (2%) of water.

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Sealed Lyophilized products prevent the reabsorption of moisture,thus, it can be stored at room temperature without refrigeration, andbe protected against spoilage for many years. Preservation is possiblebecause the greatly reduced water content inhibits the action ofmicroorganisms and enzymes that would normally spoil or degrade thesubstance.

Generic name Dosage form Therapeutic category

Aceclofenac Injection 150mg Lyophillized Injection NSAID

Azithromycin for Injection

500mg/vialLyophillized Injection Antibacterial antibiotic

Esomeprazole Sodium Powder for

Injection 40mg/vialLyophillized Injection Proton pump inhibitor

Omeprazole for injection 40mg/vial Lyophillized Injection Proton pump inhibitor

Rabeprazole Sodium for Injection

20mg/vialLyophillized Injection Proton pump inhibitor

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REFERENCES

United States Pharmacopoeia (USP)

British Pharmacopoeia (BP)

Pharmaceutical Formulations by

M.E.Aulton, H.C. Ansel.page no 195-196. Meaning: Lysate-a mixture of substances formed by cell lysis.

Amebocytes: a migratory, ameboid cells found in many invertebrates

function in excretion and assimilation.

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