Quality Control of

Sterile Products

[Ms. Riffat]
 objectives
■ Discuss about Pyrogen test of parentrals

■ USP methods and requirements for test

■ Discuss about LAL test of parentrals

■ Know the mechanism of LAL test
■ Discuss the procedure and Advantages of
LAL test
■ Describe content uniformity test
 PYROGEN
■ Pyrogens- means fever producing
■ Having nature Endogenous (inside
body) Exogenous (outside body)
Exogenous pyrogens – mainly
lipopolysaccharides bacterial origin, but
not necessary
Sources of pyrogen
■ Solvent - possibly the most important
source
■ Medicament
■ Apparatus
■ Method of storage between
preparation and sterilization
Test for Pyrogen Rabbit test
■ Development of test in 1920
■ Pyrogen test introduced into USP
(1942)
■ The test consists of measuring the rise
in body temperature in healthy rabbits
by the IV injection of a sterile solution
of the test sample.
Test conditions
■ Rabbits must be healthy and mature
■ Either sex may be used
■ Must be individually housed between
20 and 23°C
■ Selection of animals (healthy, adult,
not less than 1.5 kg,…)
Contd.
■ Equipment and material used in test
(glassware, syringes, needles)
■ All materials and glass ware to be used
must be pyrogen free (by heating
at 250 °C for 30 minutes)
■ Rabbits retaining boxes (comfortable
for rabbits)
■ Thermometers (standardized position
in rectum, precision of 0.1°C)
Rabbit’s Preliminary test
(Sham Test)
■ IV injection of sterile pyrogen-free
saline solution to exclude any animal
showing an unusual response to the
trauma (shock) of injection
■ Any animal is not used in the main test
showing a temperature variation
greater than 0.6 °C
Main test
■ Group of 3 rabbits
Preparation and injection of the
product
■ Warming the product dissolving or
dilution
■ Duration of injection: NMT 4 min
■ Injected volume: NLT 0.5 ml/kg and
NMT 10 ml/kg of body mass
Procedure
■ Inject the test solution into ear vein
of 3 rabbits.
■ At no time during a 3-hour period
following injection must temp of any
single rabbit rise more than 0.6°C Or
the sum of the rise for the three
rabbits exceed 1.4 °C in order for the
sample to pass.
Interpretation of result:
■ The test is carried out on the first
group of 3 rabbits;
■ If necessary use further groups of 3
rabbits.
■ Passing or failing of products are on
the basis of summed temperature
response
REFERENCES:
■ Remington, vol.1 Theory and practice
of industrial pharmacy
■ Dispensing for pharmaceutical student,
by: cooper and guns
LAL test
■ The name of the test is also Limulus
amebocyte lysate (LAL)- a bio chemical
test.
■ Lysate[enzyme] obtained from
horseshoe crab (Limulus polyphemus
or Tachypleus tridentatus)
Mechanism of LAL test
■ Mechanism based on the primitive
blood-clotting of the horseshoe crab
enzymes as a consequence coagulation
produce proteinaceous gel
Procedure
■ Equal Volumes of LAL reagent and test
solution (usually 0.1 ml of each) are
mixed in a de-pyrogenated test-tube
■ Incubation at 37°C, after 1 hour
afterward the tube - invert in one
smooth motion (180°)
■ Observe the result pass/fail test
Test Principle :
■ The addition of lysate produce turbidity,
precipitation/gel formation of the
mixture.
■ The rate of reaction depends on the
concentration of the endotoxin.
Guidelines
■ Gel formation = test Fails
■ No gelling = test Pass
■ The absence of bacterial endotoxins in
a product implies the absence of
pyrogenic component
Advantages of LAL test
■ Fast test - 60 minutes vs. 180 minutes
■ Greater Sensitivity
■ Less Variability
■ Much Less False Positives
■ Much Less Expensive
■ Alternative to Animal Model
■ Cheaper, more accurate than other
Contd.
■ Particularly useful for: Radiopharm,
cytotoxic agents, products with marked
pharmacological or toxicological activity
in the rabbit (e.g. insulin), water for
injection where LAL test is potentially
more stringent and readily applied
UNIFORMITY OF CONTENT
■ Determine the content of API of each
of 10 containers selected randomly
using the method given in the
monograph or by any other suitable
analytical method.
■ The preparation being examined
complies the test if the potency lies 85
-115 % of the average value. [i.e. ±
15%]
REFERENCES:
■ Remington, vol.1 Theory and practice
of industrial pharmacy
■ Dispensing for pharmaceutical student,
by: cooper and guns