1. Prepared By:
Pramod Ananda Ramane,
M-Pharm, FirstYear, Roll No.10
P’ceutical Quality Assurance Department,
B.V.C.P. Kolhapur
IN PROCESS QUALITY CONTROL
TESTS FOR STERILE DOSAGE
FORMS
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2. INDEX
 Introduction to Sterile Products
IPQC
IPQCTests for Sterile Formulations
•LeakageTest
•ClarityTest
•pH
•Particulate Matter Injection
•SterilityTest
•PyrogenTest
•Content Uniformity & Weight
•Volume Filled
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3. 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.
STERILE PRODUCTS
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4.  IPQC means controlling the procedures involved in manufacturing of
the dosage forms starting from raw materials purchase to dispatch of
the quality product in ideal packaging.
 It monitors all the features of the product that may affect its quality and
prevents errors during processing.
 These are the tests performed between QA and QC and provides for the
authorization of approved raw materials for manufacturing based on
actual laboratory testing generally called as IPQC such as physical,
chemical, microbiologic and biologic tests.
 IPQC is concerned with providing accurate, specific & definite
descriptions of the procedures to be employed, from, the receipt of raw
materials to the release of the finished dosage forms.
IPQC
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5.  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.
LEAKAGE TEST
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6.  Leakage test is employed to detect incompletely sealed
ampoule so that they may be discarded.
 To test the packageintegrity.
 Package integrity reflects its ability to keep the product in and to
keep potential contamination out.
 Leakage tests are 4 types
1. Visual inspection
2. Bubble test
3. Dye test
4. Vacuum ionization
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7.  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.
a)Visual inspection
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8.  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.
b)Bubble test
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9.  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.
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10.  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.
C)Dye test
 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.
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11.  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.
d)Vacuum ionization test
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12.  The sensitivity of the method is not documented.
 This test is rapid and is non destructive test.
 However, the proteins present in test sample may be
decomposed.
 This method is used of for the lyophilized vials
in biopharmaceuticals.
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13. 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.
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14.  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
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15. pH
Checking the bulk solution, before filling for drug content, pH, color, clarity and
completeness of solution.
The pH of a formulation must be considered from following standpoint:
 The effect on the body when the solution is administered
 The effect on stability of the product
 The effect on container-closure system
pH measurement
 pH is measured by using a pH meter .
 pH meter is initially calibrated with respective buffer capsule then the pH of the
preparation is measured.
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16. •The preparations intended for parenteral use should be free from
particulate matter and should be clear when inspected visually.
•Two methods are described by USP according to the filled volume of the
product to be tested.
• For large volume parenterals (LVP's), a filtration followed by
microscopical examination procedure is used.
• For small volume parenterals (SVP's) a light obscuration based sensor
containing electronic liquid borne particle counter system is used.
• The USP standards are met if the LVP's under test contain NMT 50 particles
per ml of 10μ m, and NMT 5 particles per ml of 25μm in an effective linear
dimensional fashion.
•The USP standards are met if the SVP's under test contain NMT 10,000
particles per container of 10 μm, and NMT 1000 particles per container of
25μm in an effective spherical diameter.
PARTICULATE MATTER IN INJECTIONS
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17. Volume of solution
Particle
size ≥ 10
μm
Particle
size ≥25
μm
Small volume injections
(< 100 ml)
3000 per
container
300 per
container
Large volume injections
(> 100 ml)
12 per ml 2 per ml
Table 1: Limits for particle number as per IP, BP, EP and JP
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18. STERILITY TESTING :
Sterility can be defined as the freedom from the presence of viable microorganisms.
It is done for detecting the presence of viable forms of bacteria, fungi and
yeast in parenteral products.
The test for Sterility must be carried out under strict aseptic conditions in order
to avoid accidental contamination of the product during test.
All glassware's required for the test must be Sterile.
Sterility testing attempts to reveal the presence or absence of viable
microorganisms ina sample number of containers taken from batch of product.
Based on results obtained from testing the sample a decision is made as to the
sterility of the batch.
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19. Major factors of importance in sterility testing:
The environment in which the test is conducted
The quality of the culture conditions provided
The test method
The sample size
Environmental conditions:
Environmental conditions avoid accidental contamination of the product during
the test.
The test is carried out under aseptic conditions regular microbiological monitoring
should be carried out.
Culture conditions:
Appropriate conditions for the growth of any surviving organism should be
provided by the culture media selection.
The sampling procedure
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20. Sterility test methods :
1 Direct inoculation method
2 Membrane filtration method
[1] Membrane filtration methods
Selection of filters for membrane filtration:
Pore size of 0.45µ effectiveness established in the retention of microorganism’s
appropriate composition the size of filter discs is about 50 mm in diameter.
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21.  The first half is transferred into 100 ml of culture media meant for
fungi and incubated at 20˚ to 25 ˚c for not less than seven days.
 The other half is transferred into 100ml of fluid thioglycolate medium
and incubated at 30 to 35 ˚c for not less than 7 days.
The procedure of membrane filtration
 Sterilization of filtration system and membrane filtration of examined
solution under aseptic conditions.
 Filtration of the sample through a membrane filter having the nominal
size of 0.45µ and a diameter of 47mm.
 After filtration the membrane is removed aseptically from the metallic
holder and divided into two halves.
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22. Samples size to be taken
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23. [2] Direct inoculation method
Required quantities of liquid is removed from the test containers with a
sterile pipette /sterile syringe.
Aseptically transfer the specified volume of the material from each
container to vessel of culture medium
Mix the liquid with medium but not aerate excessively.
Incubate the inoculated media for not less than 14 days, unless otherwise
specified in
the monograph at 300c - 350c in the case of fluid thioglycolate medium and
200c - 250 c for soybean casein digest medium.
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24. When materials examined renders the medium turbid so presence / absence of
microbial growth cannot be determined readily by visual examination transfer
suitable portions of medium to fresh vessels of the same medium between 3 rd.
and 7 th day after test is started.
Continue incubation of the transfer vessel for not less than 7 additional days after
transfer and total of NLT 14 days.
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25. Interpretation of results
At the end of the incubation period the following observations are possible:
No evidence of growth; hence the preparation being examined passes the test for sterility.
If there is evidence of growth, retesting is performed using the same number of samples,
volumes to be tested and the media as in the original test. If no evidence of microbial
growth is then found, the preparation being examined passes the test for sterility.
If there is again evidence of the microbial growth then isolate and identify the organisms.
If they are not readily distinguishable from those growing in the containers of the first
test then the preparation being examined fails the test for sterility.
If they are distinguishable from the organisms of the first test then again do the test
using twice the number of samples. The preparation being examined passes the test for
sterility in case there is no evidence of microbial growth. In case there is evidence of
growth of any microorganisms in second re –test, the preparation being examined fails
the tests for sterility.
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26. PYROGEN TESTING
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.
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27.  Parenteral solutions are officially tested for the presence of
pyrogens by a biological test in which “FEVER” response of rabbits
is used as criteria.
Elimination of pyrogens:
1. Dry heat sterilization: For glass wares, metal equipments, powders,
waxes, oils, heat stable drugs.
650 o C temp - 1 min
250 o C temp - 30 min 180 o C temp -
240 min
2.Ultra filtration
3.Reverse osmosis: RO membrane is composed of cellulose acetate
phthalate/ polyamide
4.Distillation
5.Adsorption method
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28. Typesof 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)
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29. 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, Thermosestor or
similar probe) into a rectum cavity of Rabbit at the depth of 7.5 cm, the test
solution must be warmed at 37º 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.
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30. Table 2: Interpretation of Result (Rabbit Test)
 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 ºc.
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31. Bacterial endotoxin test
•LAL (Limulus Amebocyte Lysate) test is used to characterize the bacterial
endotoxin that may be present.
• The USP reference standard contains 10,000 USP endotoxins per vial. The LAL
reagent is used for gel-clot formation.
• The test is performed using stated amounts of volumes of products, standard,
positive control, negative control of endotoxin.
•The tubes are incubated at 37±1ºC FOR 60 ±2 minutes. When the tubes are inverted
at 180ºC angle, formation of firm gel confirms positive reaction.
•While formation of a viscous gel that doesn't maintain its integrity or absence of a
firm gel confirms negative reaction.
•The test is invalid if the standard endotoxin or positive product control doesn't show
end point within ± 1. Two fold dilution from label claim sensitivity of LAL reagent or
if the negative control shows gel-clot end point. 31

32. CONTENT UNIFORMITYAND 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 11 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.
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33. Table 3: 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
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34. • Quality control should be a fundamental segment of
parenteral products manufacturing.
• All of the 5 basic tests which are performed are essential and
have its own importance in parenteral production .
• All of these tests ensure that product meet its quality which has been
judged to satisfactory also.
• Each test is unique and provides detailed assessment of quality
control for parenteral products.
CONCLUSION
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35. REFERANCES
• Indian Pharmacopoeia
•United States Pharmacopoeia
•British Pharmacopoeia
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36. THANK YOU
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