Quality control tests of sterile products

Name: Muhammad Zohaib

Roll#: 70116396
Subject: Quality Control
Submitted To: Dr. Nariman Shahid
Section: 7(c)

1
Document Name
Your Company Name (C) Copyright (Print Date) All Rights Reserved
 Sterile Products:
Sterile product means a drug or nutritional substance that is free from living microorganisms and is
compounded, manipulated, or repackaged by pharmacy personnel, using aseptic technique and other quality
assurance procedures.
There are two types of sterile dosage forms
1. Parenteral preparation
2. Opthalmic formulations

Parenteral preparation:
Parenteral drug products include injections as well as implanted drugs injected through the skin or other
external boundary tissue or implanted within the body to allow direct administration of drug substances into
blood vessels, tissues organs or lesions. Injections may be in immediate or extended-release dose format.

Leakage Test:
The leaker test is intended to detect incompletely sealed ampules, so that they may be discarded. Tip sealed
ampoules are more prone to leak than pull sealed. In addition to that crack my present around seal or at the
base of ampule as a result of improper handling leakers are usually detected by producing negative pressure
within the incompletely sealed ampule usually into a vaccum chamber while those ampule are submerged
into a colored dye solution of 0.5 to 1% methylene blue. Vials and bottles are not subjected to such leaker
test because rubber closure is not rigid however bottles are often sealed while vaccum is pulled so that bottle
remains evacuated during its shelf life.

The presence of vaccum is detected by striking at the base of bottle sharply with the heel of hand to produce
typical water hammer sound. Another test is to apply a spark tester probe outside to the bottle moving form
liquid layer into air space a blue spark discharge occur is air space is evacuated.
1) Dye Bath Test:
A dye bath surrounds the test container. For a while, pressure and vacuum are used. Following its removal
from the dye bath, the container is cleaned. Then, using UV spectroscopy or visual inspection, the container
is checked for the presence of dye. It is possible to use blue, green, or yellowish-green dye. In order to enhance
capillary migration through the pores, the dye test can be optimized by adding a surfactant or a low viscosity
fluid to the dye solution. In the drug use industry, the dye test is widely accepted and approved. The test is
low cost and doesn't require any specialized equipment to detect visual dyes. Nevertheless, the test is sluggish,
destructive, and qualitative.

Test procedure for leakage test:

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

2
Document Name
Your Company Name (C) Copyright (Print Date) All Rights Reserved
 Pyrogen Test:
Pyrogens are products of metabolism in microorganisms Gm-ve bacteria produces most potent pyrogens.
These are lipopolysacchrides chemically and heat stable and are capable of passing through bacteria retentive
filter. When these pyrogens are introduced into a body they produce a mark response of fever with body ache
and vasoconstriction within an onset of 1 hour. Basically, there are test performed to detect the presence of
pyrogens in sterile parenteral products they are
a) Rabbit Test
b) LAL Test.

a) Rabbit test:
This test basically involves the injection Sample solution which is to be tested into a Rabbits Which are used
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.

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 to initial first 3 rabbits the solution is judged to be non-pyrogenic if NMT 3 of 8 rabbits
show individual temperature rise of 0.5 degree Celsius.

b) 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 analyzed 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.

3
Document Name
Your Company Name (C) Copyright (Print Date) All Rights Reserved
 Clarity Test (Particulate matter monitoring):
Clarity testing is carried out to check the particulate matter in the sample. In this test transparent particles or
white particles observed against the black background and the black or dark particles observed against the
white background.

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:
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 purpose.
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

Sterility Test:
The tests for sterility are intended for detecting the presence of viable microorganism in pharmaceutical
preparation that is designed to be sterile. The test is based on the principle that if micro-organism are placed
in a medium that provide optimum condition of nutrition, moisture, PH, aeration, temperature, they can grow
and their presence will be indicated by the presence of turbidity in clear medium. Test for sterility may be
carried out by one of the following two methods.

Membrane Filtration Method:

Use membrane filters having a nominal pore size not greater than 0.4μm whose effectiveness to retain
microorganisms has been established. Cellulose nitrate filters, for example, are used for aqueous, oily, and
weakly alcoholic solutions; and cellulose acetate filters, for example, are used for strongly alcoholic solutions.
Specially adapted filters may be needed for certain products (e.g., for antibiotics). The technique described
below assumes that membranes about 50 mm in diameter will be used. If filters of a different diameter are
used, the volumes of the dilutions and the washings should be adjusted accordingly

4
Document Name
Your Company Name (C) Copyright (Print Date) All Rights Reserved
 The filtration apparatus and membrane are sterilized by appropriate means. The apparatus is designed so that
the solution to be examined can be introduced and filtered under aseptic conditions: it permits the aseptic
removal of the membrane for transfer to the medium, or it is suitable for carrying out the incubation after
adding the medium to the apparatus itself. After filtration the preparation membrane is cut into two halves.
One halves is transferred in to 100ml of culture medium meant for the growth of the bacteria and incubated
at 30 to 35°C for not less than 7 days. The another halve is transferred to 100 ml of culture medium meant
for fungi and incubated at 20 - 25 oC for not less than 7 days.

Direct Inoculation Method

Although international pharmacopoeias recommend using standard membrane filtration for sterility testing,
there are certain products that are not filterable or deformable. These products are normally tested using direct
inoculation. In this method, the test sample is added directly into the required media, ensuring that the amount
of sample is below 10%. To comply with your different direct inoculation method requirements, we offer
sterility test media in various volumes, from 9mL tubes up to 75 mL bottles. In this method an aliquot quantity
of the material being tested is drawn aseptically from the container and transferred to a vessel containing a
measured quantity of a suitable culture medium. The culture is incubated at appropriate temperature for not
less than 14 days. The culture medium is observed at periodic intervals during the incubation period and at
the end to detect presence of any microbial growth.

Test interpretation (criteria or limits):

If no visible evidence of microbial growth in culture medium in test tube then it is interpreted that the sample
representing lot is without intrinsic contamination. If visible microbial growth is seen or if the test is judged
to be invalid because of inadequate environmental conditions the sterility test is repeated
such interpretation must be made by those personnel who have adequate knowledge of aseptic processing,
industrial sterilization methods, and environmental control procedures used in test facility.

Content Uniformity & Weight:

Determine the content of the active ingredient of each of 10 containers taken at random. The preparation
under examination complies with the test if the individual values thus obtained are all between 85 and 115
percent of the average value. The preparation under the examination fails to comply with the test if more than
one individual value is outside the limits 85 to 115 percent of the average value or if any one individual value
is outside the limits 75 to 125 percent of the average value. If one individual value is outside the limits 85 to
115 percent but within the limits 75 to 125 percent of the average value, repeat the determination using
another 20 containers taken at random. The preparation under examination complies with the test if in the
total sample of 30 containers not more than one individual value is outside the limits 85 to 115 percent and
none is outside the limits 75 to 125 percent of the average value. Limits for uniformity of weight is given in
Table
Table 1 Limits for Uniformity of Weight

Pharmaceutical Formulation Average Mass Percentage Deviation (%)

Powders for parenteral use More than 40 mg 10
Powders for eye drops Less than 300 mg 10
Powders for eye lotions 300 mg or more 7.5

5
Document Name
Your Company Name (C) Copyright (Print Date) All Rights Reserved
 Ophthalmic preparations:
Ophthalmic preparations are sterile liquid, semi-solid or solid preparations intended for administration upon
the eyeball and/or to the conjunctiva or for insertion in the conjunctive sac.

Several categories of eye preparations may be distinguished:

1. Eye drops,
2. Eye lotions,
3. Powders for eye drops and eye lotions,
4. Semi-solid eye preparations,
5. Ophthalmic inserts.

Specific tests for ophthalmic preparations are discussed below, Test for sterility, pyrogen test, particulate
matter test, uniformity of weight, deliverable mass or volume tests are similar to that of parenteral
preparations.

Quality control test for Ophthalmic preparations:

Particle size:
Introduce a suitable quantity of the preparation into a counting cell or with a micropipette onto a slide, as
appropriate and scan under microscope an area corresponding to 10 μg of the solid phase. For practical
reasons, it is recommended that the whole sample is first scanned at low magnification (e.g. 50X) and
particles greater than 25 μm are identified. These larger particles can then be measured at a larger
magnification (e.g. 200X to 500X).
For each 10 μg of solid active substance, particle size and number of particles are given in the
Table 2 Limits for particle number as per IP, USP, BP, JP, Ph. Eur

Pharmacopoeia Particle Size No. of particles allowed

Particle
IP 25 μm Not more than 20
50 μm Not more than 2
100 μm Nil
BP, JP, Ph. Eur 5 μm Not more than 20
50 μm Not more than 10
90 μm Nil

Uniformity of content:
Determine the content of the active ingredient of each of 10 containers taken at random. The preparation
under examination complies with the test if the individual values thus obtained are all between 85 and 115
percent of the average value. The preparation under the examination fails to comply with the test if more than
one individual value is outside the limits 85 to 115 percent of the average value or if any one individual value
is outside the limits 75 to 125 percent of the average value. If one individual value is outside the limits 85 to
115 percent but within the limits 75 to 125 percent of the average value, repeat the determination using
another 20 containers taken at random. The preparation under examination complies with the test if in the
total sample of 30 containers not more than one individual value is outside the limits 85 to 115 percent and
none is outside the limits 75 to 125 percent of the average value

6
Document Name
Your Company Name (C) Copyright (Print Date) All Rights Reserved
 Uniformity of volume:
Pour completely the contents of each container into calibrated volume measures of the appropriate size and
determine the volume of contents of 10 containers. The average net volume of the contents of the 10
containers is not less than the labeled amount, and the net volume of the contents of any single container is
not less than 91% and not more than 109 % of the labeled amount where the labeled amount is 50 ml or less
or not less than 95.5% and not more than 104.5% of the labeled amount where the labeled amount is more
than 50 ml but not more than 200 ml, or not less than 97% and not more than 103% of the labeled amount
where the labeled amount is more than 200 ml but not more than 300 ml. If these requirements are not met,
determine the net volume of the contents of 10 additional containers. The average net volume of the contents
of the 20 containers is not less than the labeled amount, and the net volume of the contents of not more than
1 of the 20 containers is less than 91 % or more than 109% of the labeled amount where the labeled amount
is 50 ml or less or not less than 95.5% and not more than 104.5% of the labeled amount where the labeled
amount is more than 50 ml but not more than 200 ml, or not less than 97% and not more than 103% of the
labeled amount where the labeled amount is more than 200 ml but not more than 300 ml.

Test for metal particles in ophthalmic ointments:

Take 10 ophthalmic ointments to be tested, and extrude the contents into a Petri dish. Cover the dish and heat
between 85°C to 110°C for 2 h to dissolve the bases. Allow the sample to room temperature without agitation
to solidify the contents. Invert each dish on the stage of suitable microscope previously adjusted to provide
more than 40 times magnifications and equipped with eye piece micrometer disk. Each dish is illuminated
from above 45° relative to the plane dish. Examine the entire bottom of each dish for metal particles and
record the total number of particles, measuring 50 μm or more in dimensions. No particles should be present.

Insoluble particulate matter test for ophthalmic solutions:

Fit the membrane filter on to the membrane filter holder. Filter under reduced pressure 200 ml of the purified
water for particulate matter test at the rate of 20 to 30 ml / min. Apply vacuum until the surface of the
membrane is free from water and remove the membrane and dry it carefully below 50°C. After the filter is
dried, place it under the microscope. Adjust the microscope to get the best view of the particles that are equal
to or greater than 150 μm. Ascertain that the number is not more than 1.
Fit another membrane filter and wet it with purified water for particulate matter test. Pour the sample solution
into the filter. For viscous solutions, dilute suitably with purified water for particulate matter test and filter.
When the amount of solution on the filter becomes small, add 30 ml of water. Repeat the process 3 times
with 30 ml of the water. Apply the vacuum gently until the surface of membrane filter is free from water. Dry
it and observe under microscope. Count the number of particles which are equal to or larger than 300 μm.

Uniformity of weight:
Remove labels and wash the container and dry. Weigh the container along with its contents. Empty the
containers as completely as possible. Rinse with water and with ethanol and dry at 100°C to a constant weight.
Allow to cool in desiccators and weigh. The difference between the weights represents the weight of the
contents. Repeat the procedure with further 19 containers and determine the average weight. Not more than
two of the individual weights deviate from the average weight by more than 10% and none deviates by more
than 20%.

7
Document Name
Your Company Name (C) Copyright (Print Date) All Rights Reserved