QUALITY CONTROL OF STERILE PRODUCTS

(Parenteral)

DEFINATION:
Parenteral refers to the injectable as well as ophthalmic preparations.
It derived from Greek words Para (Outside) and enteron (Intestine). So it is a route of
administration other than the oral route. This route of administration bypasses the
alimentary canal.
These parenteral preparations must be sterile which means the products must be free of
microorganisms as well as spores. The spores are pyrogen (dead body of microorganism,
parts of microorganism) or endotoxin etc.

Pyrogens are the fever-producing substances i.e. metabolic by-products of microbial

growth, dead microorganism and part of microbial. They may be soluble, insoluble, or
colloid.
Endotoxin is lipopolysaccharides which release after lysis of gram negative bacterial cell
wall causing an increase in the body temperature.
A sterile product is one that is free from living microorganisms or viable microorganisms
which are able to reproduce and sterility conditions of the absence of micro organisms.
This condition is achieved by sterilization process.

Sterilization: is the process in which we kill or remove the microorganism that may be able to
contaminate that environment or vessel.
There are a number of different methods of sterilization. They include:
1. Heat (steam or dry heat)
2. Radiation (e.g., gamma ray or electron beam)
3. Gas (e.g., ethylene oxide)
4. Filtration (with subsequent aseptic handling by filters)

Evaluation of Sterile Products:

 Leak/Leakage test
 Clarity test
 Pyrogen testing
 LAL test
 Assay or drug content uniformity
1. LEAKER TEST FOR AMPOULES (USP):
Ampoules are small glass containers that contain a sterile medicinal liquid intended for
parenteral use. Presence of capillary pores or tiny cracks can cause microbes or other
dangerous contaminants to enter the ampoules or may lead to the leakage of contents
to outside. This may lead to contamination of the sterile contents and also spoilage of
appearance of the package.
Leaker test for ampoules is intended to detect incompletely sealed ampoules so that
they can be discarded in order to maintain the sterile conditions of the medicines. Tip
seals are more likely to be incompletely closed than pull seals. Open capillaries or
cracks at the point of seal result in LEAKERS.

TEST PROCEDURE:

1. Filled and sealed ampoules are placed in a vacuum chamber and completely
submerge/ immerse in a deeply colored dye solution of about 0.5 to 1% methylene
blue.
2. A negative pressure (vacuum) of about 27 inches Hg, for 30 minutes is applied/
created within the vacuum chamber of the leaker test.
3. On the release or removal of pressure, the dye penetrates into the ampoules through
an opening thus making it visible after the ampoule has been washed.
4. Detection of leakers is prominent when ampoules are immersed in a bath of dye
during autoclaving cycle as this has the advantage of accomplishing both leaker
detection and sterilization in one operation.
5. This test is conducted on the whole batch (100%) and all the defected ampoules
must be rejected i.e. zero tolerance.
6. Finally observe the visual defects such as cracks, pinholes, and capillaries.

 DISADVANTAGES: Capillaries of 15 micron or smaller diameter cannot be detected

by this test. Vials and bottles are not subjected to such a leaker test as the rubber
closer is not rigid.

CHEMICAL TRACER TESTS: This test follows the principle of interaction between
material i.e. ammonia on one side and hydrochloric acid on the other side which forms
a white cloud of ammonium chloride indicating leakage. Pinhole can be well detected by
this method.

THERMOCOUPLE GAUGES:
This technique based on the mechanism of temperature change. These are mainly used
to detect a drop in temperature when a solvent type systems escape under vacuum. It is
also used to detect the presence of warmer gases.
 OTHER PROCEDURES:
1. LEAKAGE TEST FOR INJECTABLE AND NON INJECTABLE PLASTIC CONTAINERS:
A) Fill about 10 containers with water and fit with an appropriate closure.
B) Keep these containers in an inverted position at room temperature for about 24
hrs. Check for any leakage from any container.

2. WATER VAPOUR PERMEABILITY TEST:

A) Take five containers and fill them with nominal volume of water.
B) Heats seal these containers with an aluminum foil - polyethylene laminate or
other seal.
C) Weigh the containers accurately and allow to stand for 14 days at a relative
humidity of 55 - 65% and a temperature between 20 to 25oC.
D) Reweigh the containers. The loss in weight in each container should not be more
than 0.2%.

REFERENCES:
Evans ER, Hall IH. Pharmaceutical Packaging Technology, 1st ed. London, Taylor &
Francis; 2000: Page-335.

2. CLARITY TEST:(Particulate matter monitoring)

Particulate matter is defined as unwanted mobile insoluble matter other than gas
bubble present in the product.
If the particle size of foreign matter is larger than the size of R.B.C.. It can block the
blood vessel.
• The permit limits of particulate matter as per USP are follows:

Indian Pharmacopeia
 Source of particulate matter:
 Intrinsic contamination: The material which are originally present in the
parenteral solution e.g. Barium ions leach in parenteral & react with sulphur ions in
the product to form barium sulphate crystals.
 Extrinsic contamination: The material which comes from the environment e.g.
Shedding of material from cloth, body, & cotton, paper, rubber, tissue etc.

 Methods for monitoring particulate matter contamination:

a) Visual method
b) Microscopic Coulter counter method
c) Filtration method
d) Light blockage method
 Identification of particulate matter:
1) Microscopy
2) X-ray powder diffraction
3) Mass spectroscopy
4) Polarized light spectroscopy
5) Scanning electron microscopy (SEM)

a) Visual method
 The containers are examined against strong illuminated screen having black and
white background.
 Black background is used for light or colorless particles and white background is
used for dark particles.
 Automatic inspection machine and manual are used for this purposes

b) Microscopic method

Membrane filters and microscope are used for this purpose. The particles retained
by the filters are then observed and count with the help of the microscope.

c) Light blockage/Obstruction method

High intensity light beam (HIAC) is used to count the particles.

d) Coulter counter Method

The sample solution is added to an electrolyte solution which is drawn through
small orifice. Voltage pulses are proportional to the particle size and the particle
below 0.2 micron can be detected
3. PYROGEN TEST

Pyrogens are the fever producing substances i.e. metabolic by-products of bacteria.
These can be either internal (endogenous) or external (exogenous) to the body.
An example of an exogenous pyrogen is called “Endotoxin” whereas endogenous
pyrogens are cytokines, molecules that are a part of the innate immune system. They
are produced by phagocytic cells and cause the increase in the thermoregulatory set-
point in the hypothalamus which causes fever, blood coagulation, and hypotension..
Examples of endogenous pyrogens are interleukin 1 (α and β), interleukin 6 (IL-6),
interleukin-8, tumor necrosis factor-α, β, macrophage inflammatory protein-α and β as
well as interferon-α, interferon-β, and interferon-γ.

Bacterial Endotoxin is the lipopolysaccharides (LPS) present in bacterial cell walls of

gram negative bacteria release after lysis. It is stable to at least 175oC and steam
sterilization is ineffective. Some endotoxins are water soluble, some are insoluble and
some are colloidal. Water soluble endotoxins i.e. monomer unit of LPS are 10,000
Daltons (1.8 nm) in size so endotoxins can easily pass through 0.22μm filters.

Sources: Pyrogens and endotoxins are found in Water (main), raw materials,
equipments, process environment, people, and gram negative bacteria.

Elimination of pyrogens: Low doses of Pyrogen are asymptomatic and moderate doses
causes fever & changes in plasma composition. High doses cause cardiovascular
dysfunction, vasodilatation, vasoconstriction, endothelium dysfunction, multiple organ
failure & finally death so Depyrogenation is very important which can be achieved by;
a) Ultra Filtration,
b) Distillation,
c) Chromatography,
d) Reverse osmosis : RO membrane is composed of cellulose acetate phthalate/
polyamide
e) Inactivation by dry heat sterilization : For glass wares, metal equipments,
powders, waxes, oils, heat stable drugs are sterilized by dry heat method at a
temperature of ;
650 o C temp - 1 min
250 o C temp - 30 min
180 o C temp - 240 min
Principle of pyrogen test (USP):
The pyrogen test is designed to detect the presence of pyrogens in the preparation /
injection. The test involves measuring the rise in body temperature of rabbits following
the intravenous injection of a test solution, in a dose not to exceed 10 mL per kg body
weight injected intravenously within a period of not more than 10 minutes.

Apparatus and Diluents (USP):

a) The syringes, needles, and glassware free from pyrogens by heating at 250 for not
less than 30 minutes or by any other suitable method.
b) The washing and rinsing solution and diluents (0.9 percent of NaCl) should also be
sterile and pyrogen-free. Because the test sample is diluted with saline solution
before injected.
Temperature Recording:
a) Use an accurate temperature-sensing device such as a clinical thermometer, or
thermostat probes that have been calibrated to assure an accuracy of ±0.1oC.
b) Insert the temperature-sensing probe into the rectum of the test rabbit and, record
the rabbit's body temperature.

Test Animals:
Use 3 - 8 healthy and mature rabbits of weight not less than 1.5 kg. House the
rabbits individually in an area of uniform temperature between 20 - 23 ±3 and
free from disturbances likely to excite them.

Preliminary Test (Sham test):

a) 1 to 3 days before the test sample inject intravenously pyrogen free saline solution
to exclude any animal showing an unusual response to the trauma (shock) of
injection.
b) Record the temperature of animal at least 90 min before the injection of test sample
c) Any rabbit showing temperature of 0.6 ̊C or more is not used for main pyrogen test.
d) At least 48 hours or 2 weeks must be allowed to elapse before the animal is used
again.

Procedure for main test:

1. Perform the test in a separate area designated for them.
2. Withhold all food from the rabbits used during the period of the test. Access to
water is allowed at all times.
3. Determine the “control temperature” of each rabbit not more than 30 minutes prior
to the injection of the test dose.
4. Use only those rabbits whose ‘control temperatures’ do not vary by more than 1
from each other, and do not use any rabbit having a temperature exceeding 39.8 .
5. Unless otherwise specified in the individual monograph, inject 10ml per kg of body
weight of test solution into marginal ear vein within 10 minutes after start of
 administration. The volume of injection should not more than 10 ml/kg and should
not less than 0.5ml/kg.
6. Record the temperature at 30-minute interval. This recording should begin at least
90 min before the injection and last up to 1-3 hours after the injection of test
sample.

Test interpretation (criteria or limits):

1. Consider any temperature decreases as zero rises if no rabbit shows an individual
rise in temperature of 0.5 to its respective control temperature, the product meets
the requirements for the absence of pyrogens (Material Passed the test).
2. If any rabbit shows an individual temperature rise of 0.5 or more, continue the test
using five other rabbits. If not more than three of the eight rabbits show individual
rises in temperature of 0.5 or more and if the sum of the eight individual maximum
temperature rises does not exceed 3.3 , then the material under examination meets
the requirements for the absence of pyrogens (Material Passed the test).
USP32–NF27 Page 124

4. BACTERIAL ENDOTOXINS TEST or LAL TEST:

LAL (Limulus amoebocytes lysate) test is designed to detect or quantify bacterial

endotoxins that may be present in the test sample. The Limulus Amebocyte Lysate (LAL)
is obtained from the aqueous extracts of circulating amoebocytes of horseshoe crab
(Limulus polyphemus or Tachypleus tridentatus) which are use as a LAL Reagent.
This LAL reagent is available in the form of LAL kits.
Advantage 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
There are two methods/ techniques for this test:
1. The gel-clot techniques;
2. The photometric techniques; it is classified into two types
a) Turbidimetric method;
b) Chromogenic method,
All these method are qualitative as well as quantitative.
1. THE GEL-CLOT TECHNIQUES:
The gel-clot techniques detect or quantify endotoxins on the basis of clotting of the
LAL Reagent with endotoxin present in the test sample. The concentration of
endotoxin required to cause the lysate to clot under standard conditions is based on
the labeled sensitivity of the LAL Reagent. In this test, the reaction endpoint is
determined from dilutions by comparison with parallel dilutions of a reference
endotoxin. Quantities of endotoxin are expressed in USP Endotoxin Units (USP-
EU). [NOTE—One USP-EU is equal to one IU of endotoxin.]

Preparation of the Standard Endotoxin Stock Solution:

1. The USP Endotoxin RS (LAL kits) has a defined potency of 10,000 USP Endotoxin
Units (EU) per vial.
2. Constitute the entire contents of 1 vial of the RSE with 5 mL of LAL Reagent Water,
mix for 30 minutes, using a vortex mixer.
3. Use this concentrated solution for making appropriate serial of dilutions such as 2
, 1 , 0.5 , and 0.25 , where is concentration of endotoxin.
4. Then preserve these dilutions in a refrigerator for 14 days and Mix vigorously, using
a vortex mixer, for not less than 3 minutes before use.

Preparation of Sample Solutions:

1. Prepare sample solutions by dissolving or diluting drugs using LAL Reagent Water
or other appropriately solutions.
2. If necessary, adjust the pH of the solution in the range of 6.0 to 8.0 using an acid,
base, or suitable buffer.

Procedure:
1. Depyrogenate all glassware and other heat-stable materials in a hot-air for 30
minutes at 250 .
2. Mix a volume of the LAL Reagent (standard endotoxin solution) with an equal
volume (0.1-mL aliquots) of sample solutions in the test tube or cavity slide.
3. Incubate the reaction mixture for a period of 60 minutes at 37 ± 1 avoiding
vibration.
4. Then to observe the formation of gel, take out the tube directly from the
incubator.
 5. If a firm gel has formed then records the result as positive and if an intact gel is
not formed then the result is negative. This is a qualitative test.
6. The endpoint is the last positive test in the series of decreasing concentrations
of endotoxin.
7. Calculate the mean value of the logarithms of the endpoint concentration and
then the antilogarithm of the mean value using the following equation:
Geometric Mean Endpoint Concentration = antilog (Se / f)
Where Se is the sum of the log endpoint concentrations of the dilution series
used, and f is the number of replicate test tubes.

Endotoxin Limits:
The endotoxin limit for parenteral drugs is specified in individual monographs in units
such as EU/mL, EU/mg, or EU/Unit of biological activity.
2. THE PHOTOMETRIC TECHNIQUES: it is classified into two types
a) Turbidimetric method;
b) Chromogenic method
a) Turbidimetric method:
This method is based on the development of turbidity after cleavage of an
endogenous substrate.
This technique is classified as either endpoint-turbidimetric or kinetic-turbidimetric.
i. The endpoint-turbidimetric technique is based on the quantitative relationship
between the concentration of endotoxins and the turbidity (absorbance or
transmission) of the reaction mixture at the end of an incubation period.
ii. The kinetic-turbidimetric technique is a method to measure either the onset time
needed to reach a predetermined absorbance of the reaction mixture or the rate
of turbidity development.

b) The chromogenic method:

This method is based on the development of color after cleavage of a synthetic
peptide-chromogen complex. It measures the chromophore released from a suitable
chromogenic peptide by the reaction of endotoxins with the LAL Reagent.
This technique is also classified as either endpoint-chromogenic or kinetic-
chromogenic.
i. The endpoint-chromogenic technique is based on the quantitative relationship
between the concentration of endotoxins and the release of chromophore at the
end of an incubation period.
ii. The kinetic-chromogenic technique is a method to measure either the onset time
needed to reach a predetermined absorbance of the reaction mixture or the rate of
color development.

Procedure for the Photometric Techniques

1. Prepare an endotoxin concentration of at least three of the standard endotoxin
RS.
2. Prepare Solutions A, B, and C, of the test sample.
3. Perform the test on Solutions A, B, and C, following the instructions for the LAL
Reagent used.
7. The volume ratio of sample to LAL Reagent should be same and incubation time
is approx. 60 minutes.
8. All photometric tests are carried out at the incubation temperature of 37 ± 1
which is recommended by the LAL Reagent manufacturer.

USP32–NF27 Page 93