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By: Dr.Sadia Urooj
(Pharm.D),(M.Phil. Pharmaceutics)
Assistant Professor (L.M.D.C)
QUALITY CONTROL OF
STERILE PRODUCTS
STERILE PRODUCTS
Def:
 Sterile products are the dosage form of
therapeutic agents that are free from viable
microorganisms.
 These sterile products include the following:
 1. Parenterals
 2. Ophthalmic
 3. Irrigating preparations
 Of these parenteral products are unique among
the dosage forms of the drugs because they are
injected through skin or mucous membranes into
the internal body compartment.
1.PARENTERALS
 Term derived from Greek words “Para” outside &
“Enteron” intestine.
 Parenterals are sterile solution/suspension of drug in
aqueous or oily vehicle.
 Parenteral drugs are administered directly into the
veins, muscles or under the skin, or more specialized
tissues such as the spinal cord.
 Term parenteral used for any drug/fluid whose
delivery does not utilize the alimentary canal for
 Parenteral preparations are supplied in glass ,
plastic container and prefilled syringes with
closures are made up of plastic or elastomer.
 Categories of parenteral preparations:
 Injections or infusions
 Concentrates for injections or infusions
 Powders for injection or infusions
 Gels for injections
 Implants
Unique Characteristics of Parenterals
 Sterile
 Particulate-free
 Pyrogen free
 Stable for intended use
 pH – not vary significantly
 Osmotic pressure similar to blood
2. OPHTHALMICS
 These are the sterile liquids, semisolids or solid
preparations intended for administration upon the
eyeball and/or conjunctiva in the conjunctival sac.
 Categories of ophthalmics:
 Eye drops
 Eye lotions
 Powder for eye drops
 Powders for eye lotions
 Semisolids eye preparations
 Ophthalmic inserts
3. IRRIGATING
PREPARATIONS
 These solutions are applied topically to bath open
wounds and body cavities.
 These are sterile solutions for single use only.
 Examples of irrigating solutions are
 0.9% w/v sodium chloride solution.
 Sterile water for irrigation
 Most irrigation fluids are now available in rigid
plastic bottles.
 The containers are sealed and sterilized by moist
heat.
History Of Parenteral Therapy
 1657: First recorded injection in animals
– Sir Christopher Wren
 1855: First subcutaneous injections of drugs
using hypodermic needles
– Dr. Alexander Wood
 1920s: Proof of microbial growth resulting in
infections
– Dr. Florence Seibert
 1926: inclusion in the National Formulary
 1944: Discovery of ethylene oxide(used for
sterilization)
 1946: Organization of Parenteral drug
Association
 1961: Development of laminar air flow
concept
 1965: Development of Total Parenteral
nutrition(TPN)
Routes of Parenteral
Administration
 Intradermal (I.D.)
 Subcutaneous (S.C)
 Intramuscular (I.M.)
 Intravenous (I.V.)
 Intra-arterial
 Intracardiac
 Intra-articular (joint)
 Intrasynovial (joint fluid area)
 Intraspinal, Intrathecal (spinal fluid)
Advantages Of Parenteral
Administration
 Fastest method of drug delivery
 Viable alternative
 Use for Uncooperative patients
 Nauseous patients
 Unconscious patients
 Less patient control
 For the patient who can have nothing by
mouth
 Prolonged action
 Correcting serious fluids and electrolyte
imbalance
 TPN
Disadvantages Of Parenteral
Administration
 Trained personnel
 Pain
 Difficult to reverse an administered drug’s effects
 Manufacturing and Packaging requirements
 Cost
 Needle sticks Injury
Characteristics of parentral
Preparations
 Intravenous (IV) preparations are either:
solutions (in which ingredients are dissolved)
suspensions (in which ingredients are suspended)
 Most parenteral preparations are made of
ingredients in a sterile water medium.
 Some parenteral preparations may be
oleaginous (oily).
 Parenteral IV preparations must have
chemical properties that will not:
damage vessels or blood cells
alter the chemical properties of the blood serum
 With blood, IVs must be:
iso-osmotic
Isotonic
 Human blood plasma has a pH of 7.4
slightly alkaline
parenteral IV solutions should have a pH that is
neutral (near 7)
 Characteristics of parenteral preparations that are
important to adjust:
 Tonicity,
 Osmolality,
 pH are characteristics of parenteral preparations.
 It is important that they be adjusted to be as close as
possible to the values for human blood, to prevent
damage to blood cells and organs.
Parenteral Added Substances
 Antibacterial Agents
 Antioxidants
 Buffers
 Tonicity Contributors
 Surfactants (Emulsifying, Solubilizing, Wetting
Agents)
 Suspending or Viscosity Increasing Agents
 Local anesthetics
 Inert Gases
Vehicles For Injection
 Aqueous vehicles:
– Frequently, isotonic (to blood) to which drug may be
added at
time of use.
 Water-miscible vehicles:
– portion of the vehicle in the formulation
– used primarily to effect solubility of drugs and/or
reduce
hydrolysis
– ethyl alcohol; polyethylene glycol (liquid) and
propylene glycol
 Nonaqueous vehicles:
– Fixed oils (vegetable origin, and rancid resistance)
used in hormone preparations
Types of Water for Injection
 Water for Injection USP
 Sterile Water for Injection USP (SWFI)
 Bacteriostatic Water for Injection USP
 Sterile Water for Irrigation USP
Water for Injection USP:
 Is water obtained by distillation or by reverse osmosis.
 It conforms to the standards of purified water (It can
not contain more than 10ppm of total solid and should
have a pH between 5 and 7).
 Is also free of pyrogens.
 Is used as solvent for preparation of parentral
solutions.
Sterile Water for Injection USP (SWFI):
 Is water for injection that is sterilized and packaged in
single dose container of type 1 and 2 glass.
 These containers do not exceed a capacity of 1 L.
Bacteriostatic Water for Injection USP:
 Is sterile water for injection that contains one or more
suitable antimicrobial agents.
 It also packaged in single or multiple dose container
of type 1 and 2 glass.
 These containers do not exceed the capacity of 30ml.
Sterile Water for Irrigation USP:
 Is water for injection that is sterilized and suitably
packaged.
 It contains no antimicrobial agents or other added
substances.
Classification
 Small Volume Parenterals (25-50 ml)
 Requires little or no manipulation
 Extended stability
 Little wastage
 Do not offer flexibility in quantity/concentration
 Primary uses of SVP
 Therapeutic injections
 Ophthalmic products
 Diagnostic agents including
o Diagnostic radiopharmaceuticals
o Allergenic extracts
 Large Volume Parenterals (>100ml):
 Flexible but requires manipulation
 Used for maintenance or replacement therapy
 Free of Preservatives
 Volume must not exceed 1L (except irrigation
sols)
 Clinical Utilization of LVP
 Basic nutrition
 Restoration of electrolyte balance
 Fluid replacement
 Blood and blood products drug carriers
QUALITY CONTROL
TESTING
General areas of Quality
control
 Three General Areas are:
 1. Incoming Stock:
Routine work testing
 2. Manufacturing:
Include numerable tests, reading and
observations through out the manufacturing
process
 3. Finished Products:
Sterility test, Pyrogen test, Clarity test, Leaker
test,
In process Quality control
tests
 Conductivity measurement
 Volume filled
 Temperature for heat sterilized product
 Environmental control tests
 Visual inspection
TESTS FOR PARENTERALS
Finished product Quality control tests:
 There are mainly five Quality control test for
the parenterals are performed.
 1) LEAKER TEST
 2) CLARITY TEST
 3) PYROGEN TEST
 4) STERILITY TEST
 5) CONTENT UNIFORMITY TEST
1) LEAKER TEST
 Leakage occurs when a discontinuity exists in the
wall of a package that can allow the passage of
gas under the action of a pressure or
concentration differential existing across the wall.
 Presence of capillary pores or tiny cracks can
cause microbes or other dangerous contaminants
to enter the ampoules or package or may lead to
the leakage of contents to outside. This may
cause contamination of the sterile contents and
also spoilage of appearance of the package.
 Changes in temperature during storage can
cause expansion and contraction of the ampoule
or package and thereby causing interchange of
its contents if an opening exists.
 Leaker test is employed to detect
incompletely sealed ampoule so
that they may be discarded.
 To test the package integrity.
 Package integrity reflects its ability
to keep the product in and to keep
potential contamination out.
 Leaker tests are 4 types
a) visual inspection
b) bubble test
c) dye test
d) vacuum ionization test
a) Visual inspection
 Visual inspection is the easiest leaker test method
to perform.
 The method is used for the evaluation of large
volume parenterals.
 To increase the sensitivity of the method the
visual inspection of the sample container may be
coupled with the application of vacuum to make
leakage more readily observable.
 This method is simple and inexpensive.
Disadvantage: less sensitive
Sensitivity is increased by applying
pressure/vacuum.
b)Bubble test
 The test package is submerged in liquids.
 A differential pressure is applied on the
container.
 The container is observed for bubbles.
 Sometimes, surfactant added liquid is used
for immersion of test package.
 Any leakage is evident after the application of
differential pressure as the generation of
foaming in immersion liquid.
 The method is simple and inexpensive.
 The location of the leaks can be observed in
this method.
 Generation of a differential positive pressure of 3
psi inside the vial and observation of any
leakage using magnifying glass within a
maximum test time of 15 minutes.
 However, it is relatively insensitive and the
findings are operator dependent and are
qualitative.
 The optimized conditions can be achieved
using a surfactant immersion fluid along with
the dark background and High intensity
lighting.
C)Dye test
 The test container is immersed in a dye bath.
 Vacuum and pressure is applied for
sometime.
 The container is removed from the dye bath
and washed.
 The container is then inspected for the
presence of dye either visually or by means of
UV spectroscopy.
 The dye used is usually 0.5% to 1%
methylene blue.
 The dye test can be optimized by use of a
 The dye test is widely accepted in industry
and is approved in drug use.
 The test is inexpensive and is requires no
special equipment required for visual dye
detection.
 However, the test is qualitative, destructive
and slow.
 The test is used for ampoules.
D)Vacuum ionization test
 Vacuum ionization test is useful for testing
leakage in the vials or bottled sealed
under vacuum.
 This test is used for testing of the lyophilized
products.
 High voltage, high frequency field is applied
to vials which to cause residual gas, if present
to glow.
 Glow intensity is the function of headspace
vacuum level.
 The blue glow is the indicative of vacuum
while the purple glow indicative of no vacuum.
 The sensitivity of the method is not
documented.
 This test is rapid and is non destructive test.
 However, the proteins present in the test
sample may be decomposed.
 This method is used for the lyophilized vials
of biopharmaceuticals.
2) CLARITY TEST
(PARTICLE CONTAINMENT TEST)
 Definition:
Clarity is a relative term, its mean a clear solution
having a high polish conveys to the observer that
the product is of exceptional quality and purity.
 Clarity test is carried out to check the
particulate matter in the sample.
 It is practically impossible that every unit of lot
is perfectly free from visible particulate matter
,that is, from particles that are 30 to 40
micrometer and large in size.
 PRINCIPLE:
 This test is performed to check the particulate
contamination of injections and infusions
consists of extraneous, mobile and
undissolved particles, other than gas bubbles,
unintentionally present in the solution.
 USP limits for large volume infusion
Particle size Particle limit
10 um (or) larger/ml 50
25 um (or) larger/ml 5
TYPES OF TEST:
 Particulate matter can be detected in
parenteral product by two methods,
1. Test for visible particles
2. Test for sub visible particles
1) Test for Visible particles
Visual inspection by naked eye:
 The test is intended to provide a simple
procedure for the visual assessment of the quality
of parenteral solutions as regards visible
particles.
 In visual inspection, each injectable is inspected
visually against white and black backgrounds.
 The white background aids in detection of dark
colored particles.
 The light or reflective particles will appear against
the black background.
 Some visual-enhancing aids can increase the
efficiency.
 A magnifying lens at 2.5 × magnification set at
the eye level facilitates the inspection.
 Microscopic examination enhances detection
of particulate matter in injectables.
 Visual inspection gives the qualitative
estimation of the particulate matter.
 Acceptance Standards is that each
container checked must not contain any
visible particulate matter.
2. Test for Sub visible particle
 This test is performed to check particulate
contamination of injections and infusions consists
of extraneous, mobile un-dissolved particles,
other than gas bubbles, unintentionally present in
the solutions.
 This is further divided into two methods:
 1. Method 1 ((Light Obscuration Particle Count
Test)
 2. Method 2 (Microscopic Particle Count Test)
 When examining injections and infusions for sub-
visible particles, Method 1 is preferably applied.
 But in case of preparations having reduced clarity
or increased viscosity, the test is carried out
according to Method 2. e. g. Emulsions, colloids,
and liposomal preparations.
METHOD 1 (Light obscuration particle count
test):
Principle:
 This test is based on the principle of light
blockage which allows an automatic
determination of the size of particles and the
number of particles according to size.
 Apparatus:
 An electronic particle counting system that uses a
light obstruction sensor with a suitable feeding
device is used.
 General precautions:
 The test is carried out under conditions limiting
particulate contamination, preferably in a laminar-
flow cabinet.
 Very carefully wash the glassware and filtration
equipment used, except for the membrane filters,
with a warm detergent solution and rinse with
abundant amounts of water to remove all traces
 Immediately before use, rinse the equipment from
top to bottom, outside and then inside, with
particle-free water R.
 Take care not to introduce air bubbles into the
preparation to be examined, especially when
fractions of the preparation are being transferred
to the container in which the determination is to
be carried out.
PROCEDURE:
 Mix the contents of the sample by slowly inverting
the container 20 times successively.
 Clean the outer surfaces of the container opening
using a jet of particle-free water R, avoiding any
contamination of the contents.
 Eliminate gas bubbles by appropriate measures
such as allowing to stand for 2 min or sonicating.
 For large-volume parenterals, single units are
tested.
 For small-volume parenterals less than 25mL
in volume, the contents of 10 or more units are
combined in a cleaned container to obtain a
volume of not less than 25 ml.
 Small-volume parenterals having a volume of 25
mL or more may be tested individually.
 For large-volume parenterals or for small-volume
parenterals having a volume of 25 mL or more,
fewer than 10 units may be tested, based on an
appropriate sampling plan.
 Remove 4 portions, each of not less than 5 mL,
and count the number of particles equal to or
greater than 10 μm and 25 μm.
 Calculate the mean number of particles for the
preparation to be examined.
Evaluation:
 Test 1.A – Solutions for infusion or solutions
for injection supplied in containers with a
nominal content of more than 100 mL:
 The preparation complies with the test if the
average number of particles present in the units
tested does not exceed 25 per millilitre equal to or
greater than 10 μm and does not exceed 3 per
millilitre equal to or greater than 25 μm.
 Test 1.B – Solutions for infusion or solutions for
injection supplied in containers with a nominal
content of less than 100 mL
 The preparation complies with the test if the average
number of particles present in the units tested does
not exceed 6000 per container equal to or greater
than 10 μm and does not exceed 600 per container
equal to or greater than 25 μm.
 For preparations supplied in containers with a nominal
volume of 100 mL, apply the criteria of test 1.B.
 If the average number of particles exceeds the
limits, test the preparation by the microscopic
particle count test.
 METHOD 2 (Microscopic Particle Count Test):
 Use a suitable binocular microscope, filter
assembly for retaining particulate contamination
and membrane filter for examination.
 The microscope is equipped with an ocular
micrometer calibrated with an objective
micrometer,
 a mechanical stage and, 2 suitable
illuminators.
 The ocular micrometer is a circular diameter
graticule and consists of a large circle divided by
crosshairs into quadrants, transparent and black
reference circles 10 μm and 25 μm in diameter at
100 magnifications, and a linear scale graduated
in 10 μm increments.
 The large circle is designated the graticule field of
view (GFOV).
 Mechanical stage capable of holding and
traversing the entire filtration area of the
membrane filter.
QUALITY CONTROL OF PARENTERALS,STERILE PRODUCT
 Illuminators:
 2 illuminators are required.
 an episcopic brightfield illuminator internal to the
microscope,
 the other is an external, focusable auxiliary
illuminator.
 Filter assembly:
 The filter assembly for retaining particulate
contamination consists of a filter holder made of
glass or other suitable material, and is equipped
with a vacuum source and a suitable membrane
filter.
 Membrane filter:
 The membrane filter is of suitable size, black or
dark grey in colour, non-gridded or gridded, and
1.0 μm or finer in nominal pore size.
 Precautions:
 The test is carried out in laminar-flow cabinet.
 Very carefully wash the glassware and filter
assembly used, except for the membrane
filter, with a warm detergent solution and rinse
with abundant amounts of water to remove all
traces of detergent.
 Air bubbles should not be present.
PROCEDURE:
 Mix the contents of the samples by slowly inverting
the container 20 times successively.
 Clean the outer surfaces of the container opening
using a jet of particle-free water R and remove the
closure, avoiding any contamination of the contents.
 Eliminate gas bubbles by appropriate measures such
as allowing to stand for 2 min or sonicating.
 For large-volume parenterals, single units are
tested.
 For small-volume parenterals less than 25 mL in
volume, the contents of 10 or more units are
combined in a cleaned container;
 Small-volume parenterals having a volume of 25
mL or more may be tested individually.
 Powders for parenteral administration are
constituted with particle-free water R.
 Wet the inside of the filter holder fitted with the
membrane filter with particle-free water R and
apply vacuum.
 Entire volume is filtered.
 Maintain the vacuum until the surface of the
membrane filter is free from liquid.
 Place the filter in a Petri dish and allow the filter
to air-dry.
 After the filter has been dried, place the Petri dish
on the stage of the microscope.
 Scan the entire membrane filter under the
reflected light from the illuminating device,
 And count the number of particles that are equal
to or greater than 10 μm and the number of
particles that are equal to or greater than 25 μm
under the microscope.
 Then Calculate the mean number of particles for
the preparation to be examined.
Evaluation:
 Test 2.A – Solutions for infusion or
solutions for injection supplied in
containers with a nominal content of more
than 100 mL
 The preparation complies with the test if the
average number of particles present in the
units tested does not exceed 12 per millilitre
equal to or greater than 10 μm and does not
exceed 2 per millilitre equal to or greater than
25 μm.
 Test 2.B – Solutions for infusion or solutions
for injection supplied in containers with a
nominal content of less than 100 mL:
 The preparation complies with the test if the
average number of particles present in the units
tested does not exceed 3000 per container equal
to or greater than 10 μm and does not exceed
300 per container equal to or greater than 25 μm.
 For preparations supplied in containers with a
nominal volume of 100 mL, apply the criteria of
test 2.B.
3. PYROGENS TEST
PYROGENS:
 Pyrogens are fever producing substances.
 Pyro means ‘pyrexia’, Gen means ‘producing’.
 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.
 Parenteral solutions are officially tested for
the presence of pyrogens by a biological test
in which “FEVER” response of rabbits is used
as criteria.
Depyrogenation:
 Depyrogenation is the removal of pyrogen.
This is achieved by the following methods.
 Inactivation - Application of very high dry heat
(250 °C) for not less than 30 minutes is the
desired method for rendering material
pyrogen free.
Types of Pyrogen test
 For Detection and quantification of Pyrogens:
 Basically there are 2 tests performed to detect the
presence of pyrogens in sterile parenteral products
they are
1) In Vivo pyrogen test (Rabbit Test)
2) In Vitro pyrogen test(Limulus Amebocyte
Lysate Test)
1) In Vivo Pyrogen test (Rabbit Test)
PRINCIPLE:
 This test consists of measuring the rise in
body temperature evoked in rabbits by the
injection of a sterile solution of the substance
being examined.
SELECTION & PROTOCOL
SELECTION OF ANIMALS:
 Use healthy adult rabbits of any sex weighing not less
than 1.5kg.
 Feed them a well balanced diet not containing any
antibiotics.
 Not showing loss of body mass during one week
preceding the test.
 A rabbit should not be used in the pyrogen test if:
 It has been used in a negative pyrogen test in the
preceding three days or
 It has been used in the preceding three weeks in a
pyrogen test in which the substance under
examination fails to pass the test.
MATERIALS NEEDED:
(a) ANIMALS' QUARTERS:
 Keep the rabbits individually in a quiet area
with a uniform appropriate temperature.
 Carry out the test in a quiet room where there
is no risk of disturbance exciting the animals
in which the room temperature is within 3 °C
of that of the rabbits' living quarters, or
 In which the rabbits have been kept for at
least 18 h before the test.
(b) THERMOMETERS:
 The thermometer or electrical device which
indicates the temperature with a precision of
0.1 °C is used.
 and insert into the rectum of the rabbit to a
depth of about 5 cm. (B.P specification) or
7.2cm (USP specification). The depth of
insertion is constant for any rabbit in every
group.
 When an electrical device is used, it should be
inserted in the rectum of the rabbit 90 minutes
before injection of the solution to be examined
and left in position throughout the test.
(c) GLASSWARE, SYRINGES AND NEEDLES:
 All the glassware, syringes and needles must be
thoroughly washed with water and heated in a hot air
oven at 250°C for 30 minutes or at 200°C for an hour.
(d) RETAINING BOXES:
 The retaining boxes for rabbit in which the
temperature is being measured by an electrical device
should be made in such a way that the animals are
retained only by loosely fitting neck stocks, the rest of
the body remains relatively free, so the rabbit may sit
in a normal position.
 The animals must be put in box not less than one
hour before the test and remain there throughout the
test.
PRELIMINARY TEST (SHAM TEST):
 One to three days before testing the product, inject
pyrogen free isotonic NaCl solution (10ml/kg body
weight warmed at 38.5°C intravenously) into animal,
which has not been used during the two previous
weeks.
 Record the temperature of animal, beginning at least
90 minutes before injection and continuing for 3 hours
after injection of solution.
 Any animal showing a temperature difference greater
than 0.6°C must not be used in the main test.
MAIN TEST:
 Carry out the test using a group of three
rabbits.
PREPARATION AND INJECTION OF
SAMPLE
 Warm the liquids to be examined to
approximately 38.5°C before injection. The
sample liquid to be injected may be diluted
with a pyrogen free isotonic NaCl solution.
 Inject the solution slowly into the marginal
vein of the ear of each rabbit over a period of
four minutes, unless otherwise mentioned in
the monograph.
DETERMINATION OF INITIAL AND MAXIMUM
TEMPERATURE
 The initial temperature of each rabbit is the mean
of two temperature readings, recorded for that
rabbit at an interval of 30 minutes in the 40min
immediately preceding the injection.
 While the maximum temperature is the highest
temperature recorded for that rabbit three hours
after the injection of the preparation being tested.
 Record the temperature of each animal at an
interval of not more than 30 minutes, beginning at
least 90 minutes before the injection of the
product to be examined and continuing 3 h after
the injection.
 The difference between the initial temperature
and the maximum temperature of each rabbit is
taken to be its response.
 When this difference is negative, the result is
counted as zero response.
REJECT THE RABBIT IF:
 Rabbits showing a temperature variation greater than
0.2 °C between two successive readings in the
determination of the initial temperature are withdrawn
from the test.
 All rabbits having an initial temperature higher than
39.8 °C or less than 38.0 °C are withdrawn from the
test.
INTERPRETATION OF RESULTS
 Having carried out the test on a group of three rabbits,
repeat if necessary on further groups of three rabbits
to a total of four groups.
 If the summed response of the first group does not
exceed the figure given in the second column of the
Table, the substance passes the test.
 If the summed response exceeds the figure given in
the second column of the table but does not exceed
the figure given in the third column of the table, repeat
the test as indicated above.
 If the summed response exceeds the figure given in
the third column of the table, the product fails the test.
Depending on the results obtained tabulate the results in the following manner.
NUMBER
OF
RABBITS
MATERIAL PASSED IF
SUM OF RESPONSE
DOES NOT EXCEED.
MATERIAL FAILED IF
SUM OF RESPONSE
EXCEEDS.
3 RABBITS 1.15C 2.65C
6 RABBITS 2.80C 4.30C
9 RABBITS 4.45C 5.95C
12 RABBITS 6.10C 7.60C
 Sometimes the difference of initial and the
final temperature is negative. If the difference
is negative, the result of the rabbit test is
counted as zero response and the sample is
considered apyrogenic.
 Rabbits used in a test for pyrogens where the
mean rise in the rabbits' temperature has
exceeded 1.2 °C are permanently excluded.
Advantages of Rabbit Test
 The human and rabbits are equally
responsive to threshold levels of the pyrogens
2) In Vitro Pyrogen test
(Limulus Amebocyte Lysate, LAL Test)
 The limulus amebocyte lysate test is also called as
in-vitro pyrogen test (USP XXI Specified new test).
 Officially it is termed as bacterial endotoxin test (BET)
used to detect or quantify endotoxins from gram
negative bacteria.
 The test principle is based on the clotting of lysate of
amebocyte (an enzyme obtained from the horse shoe
crab) in the presence of pyrogens.
 The extract from the blood cells of horse shoe crab,
Limulus Polyphemus contains an enzyme system
called "Limulus- Amebocyte Lysate" (LAL) which
reacts with pyrogens so that an assay mixture
increases in viscosity and opacity until an opaque gel
is formed.
 Amebocyte + Pyrogen ~ Opaque gel
 The reaction accomplishes within 15-60 minutes,
depending on concentration of pyrogens after
mixing. The concentrated pyrogens make the gel
more turbid and thick.
REQUIREMENTS:
 Limulus-Ambocyte Lysate is prepared by bleeding
healthy mature specimens by heart puncture.
 The amebocytes are carefully concentrated,
washed and lysed by osmotic effects.
 Prior to perform the LAL test, lysate assay is
carried out with purified endotoxins and are
accepted if it detects 0.001ug/ml or less
concentration of the purified endotoxins.
 The glassware, such as glass test tubes (10 x
75mm) used in the test must be thoroughly
cleaned, dry and heat sterilized.
 A buffer solution of potassium phosphate
2mEq/ml is used to adjust the pH of test
sample at 7.
 The alcoholic content in sample is to be
removed as it causes precipitation of lysate. If
the sample contains proteins, it produces gel
thus the proteins must be diluted to
appropriate concentration before the test.
 Similarly other interfering substances present
in sample must also be removed before the
test.
PROCEDURE:
 The pH of test sample if specified is adjusted.
 The test solution and standardized LAL are
separately mixed in equal parts (0.05-0.2ml).
 The mixture is incubated immediately at 36-
38°C for 1 hour in assay tube.
 The assay tube must be remained
undisturbed completely because agitation
may irreversibly destroy the gel leading to a
false negative result.
 The test tube is observed after the specified
time and is examined for the formation of
opaque gel.
 Formation of gel represents a positive test
endpoint reaction.
 The test is performed using a commercial LAL
test kit.
 This kit contains a lyophilized LAL, and E. coli
endotoxin and pure water as standards and
these later two are used to check the
sensitivity of the test.
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( as little as 0.1ml of
test solution).
 7. It is more accurate.
4) Uniformity of contents
 The test for uniformity of content of single-dose
preparations is based on the assay of the
individual contents of active substance(s) of a
number of single-dose units to determine whether
the individual contents are within limits set with
reference to the average content of the sample.
 The test is not required for multivitamin and trace-
element preparations and in other justified and
authorised circumstances.
METHOD:
 Using a suitable analytical method, determine the
individual contents of active substance(s) of 10
dosage units taken at random.
TEST A:
 Criteria for Tablets, powders for parenteral
administration, ophthalmic inserts, suspensions
for injection:
 The preparation complies with the test if each
individual content is between 85 per cent and 115
percent of the average content.
 The preparation fails to comply with the test if more
than one
individual content is outside these limits or if one
individual content is outside the limits of 75 per cent to
 If one individual content is outside the limits of 85
per cent to 115 percent but within the limits of 75
per cent to 125 percent, determine the individual
contents of another 20 dosage units taken at
random.
 The preparation complies with the test if not more
than one of the individual contents of the 30 units
is outside 85 per cent to 115 percent of the
average content and none is outside the limits of
75 percent to 125 per cent of the average
content.
5) TEST FOR STERILITY
 The test is applied to substances, preprations or
articles which, according to pharmacopiea, are
required to be sterile.
 However, a satisfactory result only indicates that
no contaminating microorganisms has been
found in the sample examined in the conditions of
the test.
PRECAUTIONS:
 The test for sterility is carried out under aseptic
conditions.
 The working conditions in which the tests are
performed are regularly monitored by appropriate
Culture media & incubation
temperature
 The following culture media have been found to
be suitable for test of sterility.
 Fluid thioglcollate medium:
 is primarily intended for culture of anaerobic
bacteria.
 Can also detect aerobic bacteria.
 Soya bean casein digest medium:
 Is suitable for culture of both fungi and aerobic
bacteria.
FLUID THIOGLCOLLATE MEDIUM:
 Composition:
 L. Cystine 0.5g
 Sodium chloride 0.75g
 Glucose monohydrate 5.5g
 Yeast extract 5g
 Pancreatic digest of casein 15g
 Sodium thioglycollate 0.5g
 Resazurin sodium soln. 1ml, freshly prepared
 Water R 1000ml
 pH after sterilisation 7.1 ± 0.2
 Prepare the medium and sterilise using a
validation process.
 If medium is stored, store a temperature between
2˚ C to 25˚ C in a sterile, airtight container.
 Do not use medium for a longer storage period.
 Fluid thioglcollate medium is incubated at 30 – 35
˚ C.
SOYA BEAN CASEIN DIGEST MEDIUM:
 Composition:
 Pancreatic digest of casein 17.0 g
 Papaic digest of casein 3g
 Sodium chloride 5.0 g
 Dipotassium hydrogen phosphate 2.5g
 Glucose monohydrate 2.5g
 Water R 1000ml
 pH after sterilisation 7.3 ± 0.2
 Prepare and sterilise the medium.
 If medium is stored, store a temperature between
2˚ C to 25˚ C in a sterile, well closed container.
 Do not use medium for a longer storage period.
 Soya bean casein digest medium is incubated at
20 – 25 ˚ C.
 Sterility of medium:
 The media used comply with the following tests,
carried out before or in parallel with the test on
the product to be examined.
 Incubate portions of media for 14 days.
 No growth of microorganisms occur.
Growth promotion test of aerobes,
anaerobes and fungi
 Perform to check the suitability of medium for
sterility test.
 Test each batch of medium.
 Inoculate portions of thioglycolate medium with a
small no. (not more than 100CFU) of following
microorganisms;
 Clostridium sporogenes
 Pseudomonas aeruginnosa
 Staphylococcus aureus
 Use separate portion of media for each specie of
microorganisms.
 Inoculate portions of soya bean casein digest
medium with a small no. (not more than 100CFU)
of following microorganisms;
 Aspergillus brasiliensis
 Bacillus subtilis
 Candida albicans
 Use separate portion of media for each specie of
microorganisms.
 Incubate for not more than 3 days in the case of
bacteria.
 Not more than 5 days in the case of fungi.
 RESULT: The media are suitable if a clearly
visible growth of microorganisms occur.
BACTERIOSTASIS &
FUNGISTASIS
 In addition to the foregoing medium tests, prior to
conducting a sterility test on a product, determine its
level of bacteriostatic and fungistatic activity by the
following procedure:
 To each of several vessels containing the specified
quantity (15, 40, 80ml) of appropriate test medium,
add the specified quantity of product.
 Inoculate these vessels of product-medium mixtures
and the control vessels of medium with dilute culture
of bacteria and fungi that are sensitive to the product
being tested, including spores of aerobic and
anaerobic bacilli.
 Incubate all vessels at an appropriate temp. for not
less than 7 days.
 If growth of the test organisms is comparable in
control vessels and in product-medium mixture
vessels, the product is not bacteriostatic or
fungistatic.
 If the product is fungstatic or bacteriostatic,
 Either use a suitable sterile inactivating agent or
 Diluting the product with sufficient quantity of
culture medium.
Test procedure for sterility of the
product
 The test may be carried out using the technique
of membrane filtration or by direct inoculation of
the culture media with the product to be
examined.
 OPENING CONTAINERS:
 Clean the exterior surface of ampules and
closures of vials and bottles with antimicrobial
agents and make access to the contents in a
suitable manner.
 SAMPLING:
 For each unit, use not less than the volume of
product and medium specified in BP.
1. Direct inoculation of culture medium:
 Transfer the specified quantity of the preparation
to be examined directly in to culture medium so
that the volume of the product is not more than
10% of the volume of the medium.
 If the product has antimicrobial activity, carry out
test after neutralizing this with a suitable
neutralizing substance or by diluting in a sufficient
quantity of culture medium.
 When it is necessary to use a large volume of the
product it may be preferable to use a
concentrated medium.
2. Membrane Filtration:
 The technique of membrane filtration is used
whenever the nature of the product permits, that
is,
 For filterable aqueous preparation
 For alcoholic or oily preparations
 For preparations miscible with or soluble in
aqueous or oily solvents provided these solvents
do not have antimicrobial activity.
 Use membrane filters having a nominal pore size
of not greater than 0.45um with effectiveness to
retain microorganisms.
 For example:
 Cellulose nitrate filters are used for aqueous, oily and
weakly alcoholic solutions.
 Cellulose acetate filters are used for strongly
alcoholic solutions.
 Special filters may be needed for certain products
.e.g. Antibiotics.
 The diameter of membrane is about 50mm.
 The filtration apparatus and membrane are sterilized
under aseptic conditions.
 If appropriate, transfer a small quantity of a suitable,
sterile diluent such as a 1 g/L neutral soln. Of meat or
casein peptone on to membrane in the apparatus and
filter.
 Transfer the contents of the container to be tested
to the membrane.
 Filter immediately.
 If the product has antimicrobial activity, wash the
membrane not less than 3 times by filtering
through it the volume of chosen sterile diluent.
 Transfer the whole membrane to the culture
medium or cut it aseptically into equal parts and
transfer one half to each of suitable media.
 Alternately transfer the medium on to the
membrane in the apparatus.
 Incubate the medium for not less than 14 days.
OBSERVATIONS &
INTERPRETATION OF RESULTS
 At intervals during the incubation and at its
conclusion, examine media for microbial growth.
 If the material being tested rendered the medium
turbid, so that the presence or absence of
microbial growth can not be readily determined by
visual inspection.
 In this case, 14 days after the beginning of
incubation transfer portion of the medium not less
than 1ml to fresh vessels of the same medium
and then incubate the original and transfer
vessels not less than 4 days.
 If no evidence of microbial growth is found, the
product to be examined complies with the test for
sterility.
 If evidence of microbial growth is found, the
product to be examined does not complies with
the test for sterility.
 When using the technique of membrane filtration ,
use whenever possible, the whole contents of
container but not less than specified quantity.
 When the volume or the quantity of in a single
container is insufficient to carryout test, the
contents of 2 or more containers are used to
inoculate different media.

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QUALITY CONTROL OF PARENTERALS,STERILE PRODUCT

  • 1. By: Dr.Sadia Urooj (Pharm.D),(M.Phil. Pharmaceutics) Assistant Professor (L.M.D.C) QUALITY CONTROL OF STERILE PRODUCTS
  • 2. STERILE PRODUCTS Def:  Sterile products are the dosage form of therapeutic agents that are free from viable microorganisms.  These sterile products include the following:  1. Parenterals  2. Ophthalmic  3. Irrigating preparations  Of these parenteral products are unique among the dosage forms of the drugs because they are injected through skin or mucous membranes into the internal body compartment.
  • 3. 1.PARENTERALS  Term derived from Greek words “Para” outside & “Enteron” intestine.  Parenterals are sterile solution/suspension of drug in aqueous or oily vehicle.  Parenteral drugs are administered directly into the veins, muscles or under the skin, or more specialized tissues such as the spinal cord.  Term parenteral used for any drug/fluid whose delivery does not utilize the alimentary canal for
  • 4.  Parenteral preparations are supplied in glass , plastic container and prefilled syringes with closures are made up of plastic or elastomer.  Categories of parenteral preparations:  Injections or infusions  Concentrates for injections or infusions  Powders for injection or infusions  Gels for injections  Implants
  • 5. Unique Characteristics of Parenterals  Sterile  Particulate-free  Pyrogen free  Stable for intended use  pH – not vary significantly  Osmotic pressure similar to blood
  • 6. 2. OPHTHALMICS  These are the sterile liquids, semisolids or solid preparations intended for administration upon the eyeball and/or conjunctiva in the conjunctival sac.  Categories of ophthalmics:  Eye drops  Eye lotions  Powder for eye drops  Powders for eye lotions  Semisolids eye preparations  Ophthalmic inserts
  • 7. 3. IRRIGATING PREPARATIONS  These solutions are applied topically to bath open wounds and body cavities.  These are sterile solutions for single use only.  Examples of irrigating solutions are  0.9% w/v sodium chloride solution.  Sterile water for irrigation  Most irrigation fluids are now available in rigid plastic bottles.  The containers are sealed and sterilized by moist heat.
  • 8. History Of Parenteral Therapy  1657: First recorded injection in animals – Sir Christopher Wren  1855: First subcutaneous injections of drugs using hypodermic needles – Dr. Alexander Wood  1920s: Proof of microbial growth resulting in infections – Dr. Florence Seibert
  • 9.  1926: inclusion in the National Formulary  1944: Discovery of ethylene oxide(used for sterilization)  1946: Organization of Parenteral drug Association  1961: Development of laminar air flow concept  1965: Development of Total Parenteral nutrition(TPN)
  • 10. Routes of Parenteral Administration  Intradermal (I.D.)  Subcutaneous (S.C)  Intramuscular (I.M.)  Intravenous (I.V.)  Intra-arterial  Intracardiac  Intra-articular (joint)  Intrasynovial (joint fluid area)  Intraspinal, Intrathecal (spinal fluid)
  • 11. Advantages Of Parenteral Administration  Fastest method of drug delivery  Viable alternative  Use for Uncooperative patients  Nauseous patients  Unconscious patients  Less patient control  For the patient who can have nothing by mouth  Prolonged action  Correcting serious fluids and electrolyte imbalance  TPN
  • 12. Disadvantages Of Parenteral Administration  Trained personnel  Pain  Difficult to reverse an administered drug’s effects  Manufacturing and Packaging requirements  Cost  Needle sticks Injury
  • 13. Characteristics of parentral Preparations  Intravenous (IV) preparations are either: solutions (in which ingredients are dissolved) suspensions (in which ingredients are suspended)  Most parenteral preparations are made of ingredients in a sterile water medium.  Some parenteral preparations may be oleaginous (oily).  Parenteral IV preparations must have chemical properties that will not: damage vessels or blood cells alter the chemical properties of the blood serum
  • 14.  With blood, IVs must be: iso-osmotic Isotonic  Human blood plasma has a pH of 7.4 slightly alkaline parenteral IV solutions should have a pH that is neutral (near 7)  Characteristics of parenteral preparations that are important to adjust:  Tonicity,  Osmolality,  pH are characteristics of parenteral preparations.  It is important that they be adjusted to be as close as possible to the values for human blood, to prevent damage to blood cells and organs.
  • 15. Parenteral Added Substances  Antibacterial Agents  Antioxidants  Buffers  Tonicity Contributors  Surfactants (Emulsifying, Solubilizing, Wetting Agents)  Suspending or Viscosity Increasing Agents  Local anesthetics  Inert Gases
  • 16. Vehicles For Injection  Aqueous vehicles: – Frequently, isotonic (to blood) to which drug may be added at time of use.  Water-miscible vehicles: – portion of the vehicle in the formulation – used primarily to effect solubility of drugs and/or reduce hydrolysis – ethyl alcohol; polyethylene glycol (liquid) and propylene glycol  Nonaqueous vehicles: – Fixed oils (vegetable origin, and rancid resistance) used in hormone preparations
  • 17. Types of Water for Injection  Water for Injection USP  Sterile Water for Injection USP (SWFI)  Bacteriostatic Water for Injection USP  Sterile Water for Irrigation USP
  • 18. Water for Injection USP:  Is water obtained by distillation or by reverse osmosis.  It conforms to the standards of purified water (It can not contain more than 10ppm of total solid and should have a pH between 5 and 7).  Is also free of pyrogens.  Is used as solvent for preparation of parentral solutions. Sterile Water for Injection USP (SWFI):  Is water for injection that is sterilized and packaged in single dose container of type 1 and 2 glass.  These containers do not exceed a capacity of 1 L.
  • 19. Bacteriostatic Water for Injection USP:  Is sterile water for injection that contains one or more suitable antimicrobial agents.  It also packaged in single or multiple dose container of type 1 and 2 glass.  These containers do not exceed the capacity of 30ml. Sterile Water for Irrigation USP:  Is water for injection that is sterilized and suitably packaged.  It contains no antimicrobial agents or other added substances.
  • 20. Classification  Small Volume Parenterals (25-50 ml)  Requires little or no manipulation  Extended stability  Little wastage  Do not offer flexibility in quantity/concentration  Primary uses of SVP  Therapeutic injections  Ophthalmic products  Diagnostic agents including o Diagnostic radiopharmaceuticals o Allergenic extracts
  • 21.  Large Volume Parenterals (>100ml):  Flexible but requires manipulation  Used for maintenance or replacement therapy  Free of Preservatives  Volume must not exceed 1L (except irrigation sols)  Clinical Utilization of LVP  Basic nutrition  Restoration of electrolyte balance  Fluid replacement  Blood and blood products drug carriers
  • 23. General areas of Quality control  Three General Areas are:  1. Incoming Stock: Routine work testing  2. Manufacturing: Include numerable tests, reading and observations through out the manufacturing process  3. Finished Products: Sterility test, Pyrogen test, Clarity test, Leaker test,
  • 24. In process Quality control tests  Conductivity measurement  Volume filled  Temperature for heat sterilized product  Environmental control tests  Visual inspection
  • 25. TESTS FOR PARENTERALS Finished product Quality control tests:  There are mainly five Quality control test for the parenterals are performed.  1) LEAKER TEST  2) CLARITY TEST  3) PYROGEN TEST  4) STERILITY TEST  5) CONTENT UNIFORMITY TEST
  • 26. 1) LEAKER TEST  Leakage occurs when a discontinuity exists in the wall of a package that can allow the passage of gas under the action of a pressure or concentration differential existing across the wall.  Presence of capillary pores or tiny cracks can cause microbes or other dangerous contaminants to enter the ampoules or package or may lead to the leakage of contents to outside. This may cause contamination of the sterile contents and also spoilage of appearance of the package.  Changes in temperature during storage can cause expansion and contraction of the ampoule or package and thereby causing interchange of its contents if an opening exists.
  • 27.  Leaker test is employed to detect incompletely sealed ampoule so that they may be discarded.  To test the package integrity.  Package integrity reflects its ability to keep the product in and to keep potential contamination out.  Leaker tests are 4 types a) visual inspection b) bubble test c) dye test d) vacuum ionization test
  • 28. a) Visual inspection  Visual inspection is the easiest leaker test method to perform.  The method is used for the evaluation of large volume parenterals.  To increase the sensitivity of the method the visual inspection of the sample container may be coupled with the application of vacuum to make leakage more readily observable.  This method is simple and inexpensive. Disadvantage: less sensitive Sensitivity is increased by applying pressure/vacuum.
  • 29. b)Bubble test  The test package is submerged in liquids.  A differential pressure is applied on the container.  The container is observed for bubbles.  Sometimes, surfactant added liquid is used for immersion of test package.  Any leakage is evident after the application of differential pressure as the generation of foaming in immersion liquid.  The method is simple and inexpensive.  The location of the leaks can be observed in this method.
  • 30.  Generation of a differential positive pressure of 3 psi inside the vial and observation of any leakage using magnifying glass within a maximum test time of 15 minutes.  However, it is relatively insensitive and the findings are operator dependent and are qualitative.  The optimized conditions can be achieved using a surfactant immersion fluid along with the dark background and High intensity lighting.
  • 31. C)Dye test  The test container is immersed in a dye bath.  Vacuum and pressure is applied for sometime.  The container is removed from the dye bath and washed.  The container is then inspected for the presence of dye either visually or by means of UV spectroscopy.  The dye used is usually 0.5% to 1% methylene blue.  The dye test can be optimized by use of a
  • 32.  The dye test is widely accepted in industry and is approved in drug use.  The test is inexpensive and is requires no special equipment required for visual dye detection.  However, the test is qualitative, destructive and slow.  The test is used for ampoules.
  • 33. D)Vacuum ionization test  Vacuum ionization test is useful for testing leakage in the vials or bottled sealed under vacuum.  This test is used for testing of the lyophilized products.  High voltage, high frequency field is applied to vials which to cause residual gas, if present to glow.  Glow intensity is the function of headspace vacuum level.  The blue glow is the indicative of vacuum while the purple glow indicative of no vacuum.
  • 34.  The sensitivity of the method is not documented.  This test is rapid and is non destructive test.  However, the proteins present in the test sample may be decomposed.  This method is used for the lyophilized vials of biopharmaceuticals.
  • 35. 2) CLARITY TEST (PARTICLE CONTAINMENT TEST)  Definition: Clarity is a relative term, its mean a clear solution having a high polish conveys to the observer that the product is of exceptional quality and purity.  Clarity test is carried out to check the particulate matter in the sample.  It is practically impossible that every unit of lot is perfectly free from visible particulate matter ,that is, from particles that are 30 to 40 micrometer and large in size.
  • 36.  PRINCIPLE:  This test is performed to check the particulate contamination of injections and infusions consists of extraneous, mobile and undissolved particles, other than gas bubbles, unintentionally present in the solution.  USP limits for large volume infusion Particle size Particle limit 10 um (or) larger/ml 50 25 um (or) larger/ml 5
  • 37. TYPES OF TEST:  Particulate matter can be detected in parenteral product by two methods, 1. Test for visible particles 2. Test for sub visible particles
  • 38. 1) Test for Visible particles Visual inspection by naked eye:  The test is intended to provide a simple procedure for the visual assessment of the quality of parenteral solutions as regards visible particles.  In visual inspection, each injectable is inspected visually against white and black backgrounds.  The white background aids in detection of dark colored particles.  The light or reflective particles will appear against the black background.
  • 39.  Some visual-enhancing aids can increase the efficiency.  A magnifying lens at 2.5 × magnification set at the eye level facilitates the inspection.  Microscopic examination enhances detection of particulate matter in injectables.  Visual inspection gives the qualitative estimation of the particulate matter.  Acceptance Standards is that each container checked must not contain any visible particulate matter.
  • 40. 2. Test for Sub visible particle  This test is performed to check particulate contamination of injections and infusions consists of extraneous, mobile un-dissolved particles, other than gas bubbles, unintentionally present in the solutions.  This is further divided into two methods:  1. Method 1 ((Light Obscuration Particle Count Test)  2. Method 2 (Microscopic Particle Count Test)
  • 41.  When examining injections and infusions for sub- visible particles, Method 1 is preferably applied.  But in case of preparations having reduced clarity or increased viscosity, the test is carried out according to Method 2. e. g. Emulsions, colloids, and liposomal preparations. METHOD 1 (Light obscuration particle count test): Principle:  This test is based on the principle of light blockage which allows an automatic determination of the size of particles and the number of particles according to size.
  • 42.  Apparatus:  An electronic particle counting system that uses a light obstruction sensor with a suitable feeding device is used.  General precautions:  The test is carried out under conditions limiting particulate contamination, preferably in a laminar- flow cabinet.  Very carefully wash the glassware and filtration equipment used, except for the membrane filters, with a warm detergent solution and rinse with abundant amounts of water to remove all traces
  • 43.  Immediately before use, rinse the equipment from top to bottom, outside and then inside, with particle-free water R.  Take care not to introduce air bubbles into the preparation to be examined, especially when fractions of the preparation are being transferred to the container in which the determination is to be carried out. PROCEDURE:  Mix the contents of the sample by slowly inverting the container 20 times successively.
  • 44.  Clean the outer surfaces of the container opening using a jet of particle-free water R, avoiding any contamination of the contents.  Eliminate gas bubbles by appropriate measures such as allowing to stand for 2 min or sonicating.  For large-volume parenterals, single units are tested.  For small-volume parenterals less than 25mL in volume, the contents of 10 or more units are combined in a cleaned container to obtain a volume of not less than 25 ml.  Small-volume parenterals having a volume of 25 mL or more may be tested individually.
  • 45.  For large-volume parenterals or for small-volume parenterals having a volume of 25 mL or more, fewer than 10 units may be tested, based on an appropriate sampling plan.  Remove 4 portions, each of not less than 5 mL, and count the number of particles equal to or greater than 10 μm and 25 μm.  Calculate the mean number of particles for the preparation to be examined.
  • 46. Evaluation:  Test 1.A – Solutions for infusion or solutions for injection supplied in containers with a nominal content of more than 100 mL:  The preparation complies with the test if the average number of particles present in the units tested does not exceed 25 per millilitre equal to or greater than 10 μm and does not exceed 3 per millilitre equal to or greater than 25 μm.
  • 47.  Test 1.B – Solutions for infusion or solutions for injection supplied in containers with a nominal content of less than 100 mL  The preparation complies with the test if the average number of particles present in the units tested does not exceed 6000 per container equal to or greater than 10 μm and does not exceed 600 per container equal to or greater than 25 μm.  For preparations supplied in containers with a nominal volume of 100 mL, apply the criteria of test 1.B.  If the average number of particles exceeds the limits, test the preparation by the microscopic particle count test.
  • 48.  METHOD 2 (Microscopic Particle Count Test):  Use a suitable binocular microscope, filter assembly for retaining particulate contamination and membrane filter for examination.  The microscope is equipped with an ocular micrometer calibrated with an objective micrometer,  a mechanical stage and, 2 suitable illuminators.
  • 49.  The ocular micrometer is a circular diameter graticule and consists of a large circle divided by crosshairs into quadrants, transparent and black reference circles 10 μm and 25 μm in diameter at 100 magnifications, and a linear scale graduated in 10 μm increments.  The large circle is designated the graticule field of view (GFOV).  Mechanical stage capable of holding and traversing the entire filtration area of the membrane filter.
  • 51.  Illuminators:  2 illuminators are required.  an episcopic brightfield illuminator internal to the microscope,  the other is an external, focusable auxiliary illuminator.  Filter assembly:  The filter assembly for retaining particulate contamination consists of a filter holder made of glass or other suitable material, and is equipped with a vacuum source and a suitable membrane filter.
  • 52.  Membrane filter:  The membrane filter is of suitable size, black or dark grey in colour, non-gridded or gridded, and 1.0 μm or finer in nominal pore size.
  • 53.  Precautions:  The test is carried out in laminar-flow cabinet.  Very carefully wash the glassware and filter assembly used, except for the membrane filter, with a warm detergent solution and rinse with abundant amounts of water to remove all traces of detergent.  Air bubbles should not be present.
  • 54. PROCEDURE:  Mix the contents of the samples by slowly inverting the container 20 times successively.  Clean the outer surfaces of the container opening using a jet of particle-free water R and remove the closure, avoiding any contamination of the contents.  Eliminate gas bubbles by appropriate measures such as allowing to stand for 2 min or sonicating.  For large-volume parenterals, single units are tested.  For small-volume parenterals less than 25 mL in volume, the contents of 10 or more units are combined in a cleaned container;
  • 55.  Small-volume parenterals having a volume of 25 mL or more may be tested individually.  Powders for parenteral administration are constituted with particle-free water R.  Wet the inside of the filter holder fitted with the membrane filter with particle-free water R and apply vacuum.  Entire volume is filtered.  Maintain the vacuum until the surface of the membrane filter is free from liquid.  Place the filter in a Petri dish and allow the filter to air-dry.
  • 56.  After the filter has been dried, place the Petri dish on the stage of the microscope.  Scan the entire membrane filter under the reflected light from the illuminating device,  And count the number of particles that are equal to or greater than 10 μm and the number of particles that are equal to or greater than 25 μm under the microscope.  Then Calculate the mean number of particles for the preparation to be examined.
  • 57. Evaluation:  Test 2.A – Solutions for infusion or solutions for injection supplied in containers with a nominal content of more than 100 mL  The preparation complies with the test if the average number of particles present in the units tested does not exceed 12 per millilitre equal to or greater than 10 μm and does not exceed 2 per millilitre equal to or greater than 25 μm.
  • 58.  Test 2.B – Solutions for infusion or solutions for injection supplied in containers with a nominal content of less than 100 mL:  The preparation complies with the test if the average number of particles present in the units tested does not exceed 3000 per container equal to or greater than 10 μm and does not exceed 300 per container equal to or greater than 25 μm.  For preparations supplied in containers with a nominal volume of 100 mL, apply the criteria of test 2.B.
  • 59. 3. PYROGENS TEST PYROGENS:  Pyrogens are fever producing substances.  Pyro means ‘pyrexia’, Gen means ‘producing’.  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.
  • 60.  Parenteral solutions are officially tested for the presence of pyrogens by a biological test in which “FEVER” response of rabbits is used as criteria. Depyrogenation:  Depyrogenation is the removal of pyrogen. This is achieved by the following methods.  Inactivation - Application of very high dry heat (250 °C) for not less than 30 minutes is the desired method for rendering material pyrogen free.
  • 61. Types of Pyrogen test  For Detection and quantification of Pyrogens:  Basically there are 2 tests performed to detect the presence of pyrogens in sterile parenteral products they are 1) In Vivo pyrogen test (Rabbit Test) 2) In Vitro pyrogen test(Limulus Amebocyte Lysate Test)
  • 62. 1) In Vivo Pyrogen test (Rabbit Test) PRINCIPLE:  This test consists of measuring the rise in body temperature evoked in rabbits by the injection of a sterile solution of the substance being examined.
  • 63. SELECTION & PROTOCOL SELECTION OF ANIMALS:  Use healthy adult rabbits of any sex weighing not less than 1.5kg.  Feed them a well balanced diet not containing any antibiotics.  Not showing loss of body mass during one week preceding the test.  A rabbit should not be used in the pyrogen test if:  It has been used in a negative pyrogen test in the preceding three days or  It has been used in the preceding three weeks in a pyrogen test in which the substance under examination fails to pass the test.
  • 64. MATERIALS NEEDED: (a) ANIMALS' QUARTERS:  Keep the rabbits individually in a quiet area with a uniform appropriate temperature.  Carry out the test in a quiet room where there is no risk of disturbance exciting the animals in which the room temperature is within 3 °C of that of the rabbits' living quarters, or  In which the rabbits have been kept for at least 18 h before the test.
  • 65. (b) THERMOMETERS:  The thermometer or electrical device which indicates the temperature with a precision of 0.1 °C is used.  and insert into the rectum of the rabbit to a depth of about 5 cm. (B.P specification) or 7.2cm (USP specification). The depth of insertion is constant for any rabbit in every group.  When an electrical device is used, it should be inserted in the rectum of the rabbit 90 minutes before injection of the solution to be examined and left in position throughout the test.
  • 66. (c) GLASSWARE, SYRINGES AND NEEDLES:  All the glassware, syringes and needles must be thoroughly washed with water and heated in a hot air oven at 250°C for 30 minutes or at 200°C for an hour. (d) RETAINING BOXES:  The retaining boxes for rabbit in which the temperature is being measured by an electrical device should be made in such a way that the animals are retained only by loosely fitting neck stocks, the rest of the body remains relatively free, so the rabbit may sit in a normal position.  The animals must be put in box not less than one hour before the test and remain there throughout the test.
  • 67. PRELIMINARY TEST (SHAM TEST):  One to three days before testing the product, inject pyrogen free isotonic NaCl solution (10ml/kg body weight warmed at 38.5°C intravenously) into animal, which has not been used during the two previous weeks.  Record the temperature of animal, beginning at least 90 minutes before injection and continuing for 3 hours after injection of solution.  Any animal showing a temperature difference greater than 0.6°C must not be used in the main test.
  • 68. MAIN TEST:  Carry out the test using a group of three rabbits. PREPARATION AND INJECTION OF SAMPLE  Warm the liquids to be examined to approximately 38.5°C before injection. The sample liquid to be injected may be diluted with a pyrogen free isotonic NaCl solution.  Inject the solution slowly into the marginal vein of the ear of each rabbit over a period of four minutes, unless otherwise mentioned in the monograph.
  • 69. DETERMINATION OF INITIAL AND MAXIMUM TEMPERATURE  The initial temperature of each rabbit is the mean of two temperature readings, recorded for that rabbit at an interval of 30 minutes in the 40min immediately preceding the injection.  While the maximum temperature is the highest temperature recorded for that rabbit three hours after the injection of the preparation being tested.  Record the temperature of each animal at an interval of not more than 30 minutes, beginning at least 90 minutes before the injection of the product to be examined and continuing 3 h after the injection.
  • 70.  The difference between the initial temperature and the maximum temperature of each rabbit is taken to be its response.  When this difference is negative, the result is counted as zero response. REJECT THE RABBIT IF:  Rabbits showing a temperature variation greater than 0.2 °C between two successive readings in the determination of the initial temperature are withdrawn from the test.  All rabbits having an initial temperature higher than 39.8 °C or less than 38.0 °C are withdrawn from the test.
  • 71. INTERPRETATION OF RESULTS  Having carried out the test on a group of three rabbits, repeat if necessary on further groups of three rabbits to a total of four groups.  If the summed response of the first group does not exceed the figure given in the second column of the Table, the substance passes the test.  If the summed response exceeds the figure given in the second column of the table but does not exceed the figure given in the third column of the table, repeat the test as indicated above.  If the summed response exceeds the figure given in the third column of the table, the product fails the test.
  • 72. Depending on the results obtained tabulate the results in the following manner. NUMBER OF RABBITS MATERIAL PASSED IF SUM OF RESPONSE DOES NOT EXCEED. MATERIAL FAILED IF SUM OF RESPONSE EXCEEDS. 3 RABBITS 1.15C 2.65C 6 RABBITS 2.80C 4.30C 9 RABBITS 4.45C 5.95C 12 RABBITS 6.10C 7.60C
  • 73.  Sometimes the difference of initial and the final temperature is negative. If the difference is negative, the result of the rabbit test is counted as zero response and the sample is considered apyrogenic.  Rabbits used in a test for pyrogens where the mean rise in the rabbits' temperature has exceeded 1.2 °C are permanently excluded. Advantages of Rabbit Test  The human and rabbits are equally responsive to threshold levels of the pyrogens
  • 74. 2) In Vitro Pyrogen test (Limulus Amebocyte Lysate, LAL Test)  The limulus amebocyte lysate test is also called as in-vitro pyrogen test (USP XXI Specified new test).  Officially it is termed as bacterial endotoxin test (BET) used to detect or quantify endotoxins from gram negative bacteria.  The test principle is based on the clotting of lysate of amebocyte (an enzyme obtained from the horse shoe crab) in the presence of pyrogens.  The extract from the blood cells of horse shoe crab, Limulus Polyphemus contains an enzyme system called "Limulus- Amebocyte Lysate" (LAL) which reacts with pyrogens so that an assay mixture increases in viscosity and opacity until an opaque gel is formed.
  • 75.  Amebocyte + Pyrogen ~ Opaque gel  The reaction accomplishes within 15-60 minutes, depending on concentration of pyrogens after mixing. The concentrated pyrogens make the gel more turbid and thick. REQUIREMENTS:  Limulus-Ambocyte Lysate is prepared by bleeding healthy mature specimens by heart puncture.  The amebocytes are carefully concentrated, washed and lysed by osmotic effects.  Prior to perform the LAL test, lysate assay is carried out with purified endotoxins and are accepted if it detects 0.001ug/ml or less concentration of the purified endotoxins.
  • 76.  The glassware, such as glass test tubes (10 x 75mm) used in the test must be thoroughly cleaned, dry and heat sterilized.  A buffer solution of potassium phosphate 2mEq/ml is used to adjust the pH of test sample at 7.  The alcoholic content in sample is to be removed as it causes precipitation of lysate. If the sample contains proteins, it produces gel thus the proteins must be diluted to appropriate concentration before the test.  Similarly other interfering substances present in sample must also be removed before the test.
  • 77. PROCEDURE:  The pH of test sample if specified is adjusted.  The test solution and standardized LAL are separately mixed in equal parts (0.05-0.2ml).  The mixture is incubated immediately at 36- 38°C for 1 hour in assay tube.  The assay tube must be remained undisturbed completely because agitation may irreversibly destroy the gel leading to a false negative result.
  • 78.  The test tube is observed after the specified time and is examined for the formation of opaque gel.  Formation of gel represents a positive test endpoint reaction.  The test is performed using a commercial LAL test kit.  This kit contains a lyophilized LAL, and E. coli endotoxin and pure water as standards and these later two are used to check the sensitivity of the test.
  • 79. 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( as little as 0.1ml of test solution).  7. It is more accurate.
  • 80. 4) Uniformity of contents  The test for uniformity of content of single-dose preparations is based on the assay of the individual contents of active substance(s) of a number of single-dose units to determine whether the individual contents are within limits set with reference to the average content of the sample.  The test is not required for multivitamin and trace- element preparations and in other justified and authorised circumstances.
  • 81. METHOD:  Using a suitable analytical method, determine the individual contents of active substance(s) of 10 dosage units taken at random. TEST A:  Criteria for Tablets, powders for parenteral administration, ophthalmic inserts, suspensions for injection:  The preparation complies with the test if each individual content is between 85 per cent and 115 percent of the average content.  The preparation fails to comply with the test if more than one individual content is outside these limits or if one individual content is outside the limits of 75 per cent to
  • 82.  If one individual content is outside the limits of 85 per cent to 115 percent but within the limits of 75 per cent to 125 percent, determine the individual contents of another 20 dosage units taken at random.  The preparation complies with the test if not more than one of the individual contents of the 30 units is outside 85 per cent to 115 percent of the average content and none is outside the limits of 75 percent to 125 per cent of the average content.
  • 83. 5) TEST FOR STERILITY  The test is applied to substances, preprations or articles which, according to pharmacopiea, are required to be sterile.  However, a satisfactory result only indicates that no contaminating microorganisms has been found in the sample examined in the conditions of the test. PRECAUTIONS:  The test for sterility is carried out under aseptic conditions.  The working conditions in which the tests are performed are regularly monitored by appropriate
  • 84. Culture media & incubation temperature  The following culture media have been found to be suitable for test of sterility.  Fluid thioglcollate medium:  is primarily intended for culture of anaerobic bacteria.  Can also detect aerobic bacteria.  Soya bean casein digest medium:  Is suitable for culture of both fungi and aerobic bacteria.
  • 85. FLUID THIOGLCOLLATE MEDIUM:  Composition:  L. Cystine 0.5g  Sodium chloride 0.75g  Glucose monohydrate 5.5g  Yeast extract 5g  Pancreatic digest of casein 15g  Sodium thioglycollate 0.5g  Resazurin sodium soln. 1ml, freshly prepared  Water R 1000ml  pH after sterilisation 7.1 ± 0.2
  • 86.  Prepare the medium and sterilise using a validation process.  If medium is stored, store a temperature between 2˚ C to 25˚ C in a sterile, airtight container.  Do not use medium for a longer storage period.  Fluid thioglcollate medium is incubated at 30 – 35 ˚ C.
  • 87. SOYA BEAN CASEIN DIGEST MEDIUM:  Composition:  Pancreatic digest of casein 17.0 g  Papaic digest of casein 3g  Sodium chloride 5.0 g  Dipotassium hydrogen phosphate 2.5g  Glucose monohydrate 2.5g  Water R 1000ml  pH after sterilisation 7.3 ± 0.2  Prepare and sterilise the medium.
  • 88.  If medium is stored, store a temperature between 2˚ C to 25˚ C in a sterile, well closed container.  Do not use medium for a longer storage period.  Soya bean casein digest medium is incubated at 20 – 25 ˚ C.  Sterility of medium:  The media used comply with the following tests, carried out before or in parallel with the test on the product to be examined.  Incubate portions of media for 14 days.  No growth of microorganisms occur.
  • 89. Growth promotion test of aerobes, anaerobes and fungi  Perform to check the suitability of medium for sterility test.  Test each batch of medium.  Inoculate portions of thioglycolate medium with a small no. (not more than 100CFU) of following microorganisms;  Clostridium sporogenes  Pseudomonas aeruginnosa  Staphylococcus aureus  Use separate portion of media for each specie of microorganisms.
  • 90.  Inoculate portions of soya bean casein digest medium with a small no. (not more than 100CFU) of following microorganisms;  Aspergillus brasiliensis  Bacillus subtilis  Candida albicans  Use separate portion of media for each specie of microorganisms.  Incubate for not more than 3 days in the case of bacteria.  Not more than 5 days in the case of fungi.  RESULT: The media are suitable if a clearly visible growth of microorganisms occur.
  • 91. BACTERIOSTASIS & FUNGISTASIS  In addition to the foregoing medium tests, prior to conducting a sterility test on a product, determine its level of bacteriostatic and fungistatic activity by the following procedure:  To each of several vessels containing the specified quantity (15, 40, 80ml) of appropriate test medium, add the specified quantity of product.  Inoculate these vessels of product-medium mixtures and the control vessels of medium with dilute culture of bacteria and fungi that are sensitive to the product being tested, including spores of aerobic and anaerobic bacilli.  Incubate all vessels at an appropriate temp. for not less than 7 days.
  • 92.  If growth of the test organisms is comparable in control vessels and in product-medium mixture vessels, the product is not bacteriostatic or fungistatic.  If the product is fungstatic or bacteriostatic,  Either use a suitable sterile inactivating agent or  Diluting the product with sufficient quantity of culture medium.
  • 93. Test procedure for sterility of the product  The test may be carried out using the technique of membrane filtration or by direct inoculation of the culture media with the product to be examined.  OPENING CONTAINERS:  Clean the exterior surface of ampules and closures of vials and bottles with antimicrobial agents and make access to the contents in a suitable manner.  SAMPLING:  For each unit, use not less than the volume of product and medium specified in BP.
  • 94. 1. Direct inoculation of culture medium:  Transfer the specified quantity of the preparation to be examined directly in to culture medium so that the volume of the product is not more than 10% of the volume of the medium.  If the product has antimicrobial activity, carry out test after neutralizing this with a suitable neutralizing substance or by diluting in a sufficient quantity of culture medium.  When it is necessary to use a large volume of the product it may be preferable to use a concentrated medium.
  • 95. 2. Membrane Filtration:  The technique of membrane filtration is used whenever the nature of the product permits, that is,  For filterable aqueous preparation  For alcoholic or oily preparations  For preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not have antimicrobial activity.  Use membrane filters having a nominal pore size of not greater than 0.45um with effectiveness to retain microorganisms.
  • 96.  For example:  Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions.  Cellulose acetate filters are used for strongly alcoholic solutions.  Special filters may be needed for certain products .e.g. Antibiotics.  The diameter of membrane is about 50mm.  The filtration apparatus and membrane are sterilized under aseptic conditions.  If appropriate, transfer a small quantity of a suitable, sterile diluent such as a 1 g/L neutral soln. Of meat or casein peptone on to membrane in the apparatus and filter.
  • 97.  Transfer the contents of the container to be tested to the membrane.  Filter immediately.  If the product has antimicrobial activity, wash the membrane not less than 3 times by filtering through it the volume of chosen sterile diluent.  Transfer the whole membrane to the culture medium or cut it aseptically into equal parts and transfer one half to each of suitable media.  Alternately transfer the medium on to the membrane in the apparatus.  Incubate the medium for not less than 14 days.
  • 98. OBSERVATIONS & INTERPRETATION OF RESULTS  At intervals during the incubation and at its conclusion, examine media for microbial growth.  If the material being tested rendered the medium turbid, so that the presence or absence of microbial growth can not be readily determined by visual inspection.  In this case, 14 days after the beginning of incubation transfer portion of the medium not less than 1ml to fresh vessels of the same medium and then incubate the original and transfer vessels not less than 4 days.
  • 99.  If no evidence of microbial growth is found, the product to be examined complies with the test for sterility.  If evidence of microbial growth is found, the product to be examined does not complies with the test for sterility.  When using the technique of membrane filtration , use whenever possible, the whole contents of container but not less than specified quantity.  When the volume or the quantity of in a single container is insufficient to carryout test, the contents of 2 or more containers are used to inoculate different media.