Evaluation of Sterile Dosage Form

Types of Parenteral
1. Based on types of packaging
a) Single dose units: ampoules, infusions and prefilled
disposable syringes.
b) Multiple dose units: multiple dose vials.

2. Based on the production and control

a) Small volume parenterals: volume < 100 ml,
b) Large volume parenterals: volume ≥ 100 ml,
In Process Quality Control Tests (IPQC) for Sterile Dosage
Form
• Quality Assurance: The planned and systematic activities
implemented in a quality system so that quality
requirements for a product or service will be fulfilled.
• Quality Control: The procedure or set of procedures
intended to ensure that a manufactured product or
performed service adheres to a defined set of quality
criteria or meets the requirements of the client or
customer.
IPQC
• IPQC means controlling the procedures involved in manufacturing
of the dosage forms starting from raw materials purchase to
dispatch (pengiriman) 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.
1. Leakage Test (visual inspection, bubble test, dye
tests and vacuum ionization test)
Leakage test is employed to test the package integrity.
Package integrity reflects its ability to keep the
product in and to keep potential contamination out”. It
is because leakage occurs when a discontinuity exists
in the wall of a package that can allow the passage of
gas under pressure or concentration differential
existing across the wall. Leakage test can be done by
dye bath test.
Dye Bath Test
• The test container is immersed in a dye bath. Vacuum
and pressure is applied for some time. 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 may be of blue, green, yellowish-green
color. The dye test can be optimized by use of a
surfactant and or a low viscosity fluid in the dye
solution to increase the capillary migration through
the pores.
• The test is used for ampoules and vials.
2. Clarity Test
Clarity testing is carried out to check the
particulate matter in the sample. In this test
transparent particles or white particles observed
against the black background and the black or
dark particles observed against the white
background.
3. 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 (sudut):
• 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.
4. Particulate matter in injections
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.
5. Sterility Test
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 in a 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.
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
• The sampling procedure
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.

Factors affecting growth of bacteria:

• Nutrition
• Moisture
• Air
• Temperature
• pH
• Light
• Osmotic pressure
• Growth inhibitors
Culture media used for sterility testing:
1. Fluid thioglycolate medium
2. Soybean casein digest medium

1. Fluid thioglycolate medium (FTM):

FTM provides both aerobic and anaerobic
environments within the same medium. FTM is an
excellent medium for the detection of bacterial
contamination.
Thioglycolate has the advantage of neutralizing the
bacteriostatic properties of mercurial preservatives.
Composition:
• L-cysteine, trypticase peptone, dextrose,
yeast extract, sodium chloride, sodium
thioglycolate, resazurin, agar, purified water,
final pH 7.1
• Specific role of some ingredients primarily
intended for the culture of anaerobic bacteria.
• Incubation of the media: 14 days at 30 -35°C
2. Soybean casein digest medium:
Soya-bean casein digest medium primarily intended for
the culture of both fungi and aerobic bacteria specific role
of some ingredients.
Incubation of the media: 14 days at 20 -25°C

Composition
Trypticase soya broth, trypticase peptone, phytone
peptone, sodium chloride, dipotassium phosphate,
dextrose, purified water, final pH 7.3
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
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.
• 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.
Advantages of membrane filtration over direct
inoculation method Greater sensitivity
• The entire contents can be tested providing an
advantage in the sterility testing of LVP and increasing
the ability to detect contamination.
• The antimicrobial agent and antimicrobial solutes in the
product sample can be eliminated by rinsing prior
transferring the filter into test tubes of media
• Thereby minimizing the incidence of false-negative test
results.
• Organisms present in an oleaginous (berminyak) product
can be separated from the product during filtration and
cultured in a more desirable aqueous medium.
Disadvantages
• This method cannot differentiate the extent
(tingkat) of contamination between units if
present because all product contents are
combined and filtered through a single filter
and cultured in single test tube.
• There exists a higher probability of inadvertent
(tidak sengaja) contamination in manual
operations.
[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 30 0c –
35 0c in the case of fluid thioglycolate medium and 20
0c – 25 0c for soybean casein digest medium.
• 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.
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 (dapat dibedakan) from those growing in
the containers of the first test then the preparation being
examined fails the test for sterility.
• If they are distinguishable (dapat dibedakan) 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.
6. Pyrogen test
• Pyrogen: “Pyro” (Greek = Fire) + “gen” (Greek = beginning).
• Fever producing, metabolic by-products of microbial growth and
death.
• Bacterial pyrogens are called “Endotoxins”. Gram negative bacteria
produce more potent endotoxins than gram + bacteria and fungi.
• Endotoxins are heat stable lipopolysaccharides (LPS) present in
bacterial cell walls, not present in cell-free bacterial filtrates.
• Stable to at least 175ºC; steam sterilization ineffective.
• Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm)
so endotoxins can easily pass through 0.22μm filters.
• Sources: Water (main), raw materials, equipment, process
environment, people, and protein expression systems if using gram
negative bacteria.
• Other Source: Equipment, Containers (Glass, plastic, metal), Solvent
and Solute.
Biological properties of endotoxin
• Pyrogen elevate the circulatory levels of inflammatory
cytokines which may be followed by fever, blood
coagulation, hypotension
• Low doses of Pyrogen: asymptomatic inflammation
reaction Moderate doses: fever & changes in plasma
composition
• High doses: cardiovascular dysfunction, vasodilation,
vasoconstriction, endothelium dysfunction, multiple
organ failure & finally death.
7. Content Uniformity and Weight

8. . Volume Filled
Volume in container
Physical and Chemical test
• Identity tests
These tests are qualitative chemical methods used to conform the
actual presence of compound for example color formation,
precipitation.
• Quality tests
These tests are the physical methods used to measure accurately the
characteristic properties of drug. For example: Absorbance, refractive
index.
• Purity tests
Purity tests are designed to estimate the level of all known and
significant impurities and contaminants in the drug substance under
evaluation. For example: Tests for clarity of solutions, Acidity, Alkalinity.
• Potency tests
Potency tests are assays that estimate the quantity of an active
ingredient in the drug.