QUALITY CONTROL OF STERILE PRODUCTS (1)

The in-process quality control test includes the leak and clarity testing. The
quality control of finished product required the pyrogen and sterility testing.
Leakage or Leaker’s 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 differs from permeation, which
is the flow of matter through the barrier itself.

Followings are the leak test methods.

A) VISUAL INSPECTION
Visual inspection is the easiest leak test method to perform. But this method
is least sensitive. 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. However, the method is insensitive, operator dependent, and
qualitative.
Sometimes, the method is used in combination with pressure and /or
temperature cycling to accelerate leakage to improve sensitivity.

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. 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. 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.

C) DYE TESTS

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 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 and vials.

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 online 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 on-line, 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.

CLARITY TESTING

Clarity testing is carried out to check the particulate matter in the sample.

Particulate matter

Matter of biological or non-biological origin and with observable length,

width, and thickness, e.g., bacteria, fungi, dust, dirt, fibers, plastic, rubber,
lint etc. It may be any matter, mixed accidentally during manufacturing in
the parenteral product which does not belong to the product. Particulate
matter may be tiny pieces of lint, glass, dust, rubber, metal fibers, hair,
microbes or unidentified and can make the product impure, unclean or unfit
for use.

Sources of particulate matter

Particulate contamination particularly of cellulose fibers, dust, cotton fibers,

hair, dandruff and loose skin from human origin as well as microbial
contamination may arise from the following main sources.
1. Material arising from the drug: undissolved substances and trace
contaminants etc.
2. Material arising from vehicle or added substances: These may include
those material not filtered out during a clarification process before to filling
the final container.
3. Materials present in the final container: Material already present in
container and which were not removed by rinsing prior to filling
4. Materials falling by chance into the final container during the filling
process
5. The container or closures which may be deposited in the produce during
sterilization, e.g. carbon black, whiting, zinc oxide and clay
6. Packaging components: Including glass, plastic, rubber, I/V
administration sets, etc.
7. Environmental contaminants: Including air, work tops, insects’ parts
8. Processing equipments: Including glass, stainless steel, rubber, or filter
fiber, etc.
9. Personnel: Including skin, hair, and clothing etc.

Particle size

Particles present in injectable are non-reactive, apyrogenic, sterilized.

However, by virtue of their size may biologically hazardous. The particulate
matter may be capable of blocking the blood vessels with severe results on
induction into body with injection.
A person with 20/20 vision under inspection conditions is able to detect
particles of size range 40 – 50 μm. However, it is universally accepted that
the particles size of 50 μm is detected visually by an unaided eye.
Particle size greater than 7 μm diameter is considered to be more
threatening. Pulmonary capillary are approximately 7 μm in diameter, thus
particle of this much size entrapped in vascular bed resulting in multiple
pulmonary infarction.

BIOLOGICAL HAZARDS REPORTED OF PARTICULATE MATTER

The particles may localize in lungs, liver, spleen and myocardial tissues and
may lead to thrombosis
Particles may lead to myocardial infarction due to embolic fibers.
Injection of solution contaminated with particulate matter causes
granulomas and emboli in lungs.
Compendial requirements
Due to having a potential for blockage of the capillaries, an injection must be
free from the visual evidence of particulate contamination. Thus, according
to the Compendial requirements, each final container of the injectable must
be inspected individually and the container must show evidence of
contamination with visible foreign material.

QUALITY CONTROL OF STERILE PRODUCTS (2)

CLARITY TESTING (DETECTION OF PARTICULATE MATTER)

Particulate matter can be detected in parenteral product by two methods,
including visual inspection and electronic particulate counting.
A) Visual methods
1) Visual inspection by naked eye
In visual inspection, each injectable is inspected visually against white and
black backgrounds. The white background aids in diction of dark colored
particles. The light or reflective particles will appear against the black back
ground. 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.

II) Automated visual inspection

The automatic systems are also called as the electron particles counter. The
electronic particles counter evaluates the particles in injectables
automatically. However, this method requires destruction of the
ampoule/container for the particle examination.
Electronic particles counting may be based on any one of the following
principles:
a) change in electrical resistance,
b) light blockages principle and
c) light scattering.
Some of the automated systems for visual particle inspection include
Autoskan, Eisai Ampoule inspection machine, Schering PDS/A-V system, etc.

Autoskan System
The Autoskan system is based on light scattering principle whereby the
particle in the path of a light source causes the scattering of light. The
scattered light is measured and provides the corresponding information
regarding the presence of particulate in the sample. This is a non-destructive
test.

EISAI AMPOULE MACHINE SYSTEM

The Eisai ampoule machine (AIM) system is based on the light blockage
principle. The particle size dimensions are determined with the shadow
created by the particle under light source. Assessment of the shadow is the
indication of the presence of particulate matter. This is also a non-
destructive test.
Schering PDS/A-V system
The Schering PDS/A-V System is based on light scattering by particle if
present in the sample. This is also a non-destructive test.

B) PARTICLE COUNT METHODS

Particle count methods are the USP specified microscopic methods, which
require the use of optical microscope and automatic microscope.

I) Optical Microscopic Method

The optical microscopic method requires magnification of 100 .10x. One
eyepiece must be equipped with graticule. A graticule have a series of circles
of different diameters, usually in a “under root 2 progression”. The graticule
is in circular diameter used to size the particulate. The micrometer is
graduated in 10 micro meter increments.

A circular diameter graticule

Ii) Automated Particle Counters
The automated particle counters are based on the light obscuration, light
scattering method and the electrical resistance methods. Coulter Counter
counts the particles in a sample based on the change in the electrical
resistance. Particle size detection limit in this instrument is from 0.1 to 1000
micrometer.
The powder sample requires pretreatment such as dispersion in an
electrolyte to form a very dilute suspension. The Suspension is usually
subjected to ultrasonic agitation to avoid particle agglomerates. A dispersant
may also be added to aid particle deagglomeration. Passage of particle
causes the change in electrical resistance in between the electrodes which is
proportional to the volume of particle. The change in resistance is converted
into voltage pulse which is amplified and processed electronically and split
into the particle size distribution into many different size-range.
Glass Tube
Orifice

Principle of coulter counter

This is a destructive test and large errors in measuring flakes and fibers are
expected.
This test is not recommended by FDA for parenterals.
Illustration for a coulter counter
High Accuracy (HIAC) Instrument
High Accuracy (HIAC) Instrument is based on light blockage principle. The
test is highly effective for counting the both solid and liquid suspended
particles. The instrument is calibrated easily and the test is recommended by
USP. This is destructive test method and is expensive.
P Light source
Digital
Value
Principle of light blockage

Met-One Climet Particle Counter

Met-One Climet Particle Counter is based on light scattering principle. The
particles are assessed and counted in the sample based on the principle of
light scattering. The instrument measures 6 particles sizes at a time and has
the excellent ability for the detection of large particle. The test is
destructive.

Depending on the instrument used, the sample may be presented as the

liquid as suspension or air suspension. The light emitted by a helium-neon
laser is incident on the sample particle. Light-particle interaction results in
scattering of light. The photo detector converts the signals corresponding
area/volume diameter of the particle.
Instrument based on light scatter principle

QUALITY CONTROL OF STERILE PRODUCTS (3)

Instruments for evaluation of particulate matter

System Working Principle Remarks
Visual Based Inspection
Autoskan Light Scattering Non-Destructive
Eisai Ampoule Inspection
(AIM) System
Light blockage (Shadow) Non-Destructive
Schering PDS/A-V System Light Scattering Non-Destructive
Electronic Particle Counters
Coulter Counter Change in Electric Resistance Destructive; Large errors in
measuring flakes and fibers;
Not recommended by FDA for parenterals
HIAC (High Accuracy Instruments)
Light Blockage Destructive; High Efficiency,
Easy calibration;
Recommended by USP;
Expensive
Met-One Climet Particle Counter
Light Scattering Destructive; Measures 6 particles sizes at a time;
Excellent large particle detection
Climet Instrument Light Obstruction Non-destructive
QUALITY CONTROL OF STERILE PRODUCTS

PYROGEN TESTING
Pyrogens produce symptoms of fever, chill, joint pain, malaise, headache
and other complaints following IV injection within 30-120 minutes which
may subside within 10-12 hours. Pyrogens are the heat stable, filterable and
soluble substances of 0.05-1.0 micrometer size and arise from microbial
contamination. Chemically these are lipopolysaccharides from the outer cell
wall of the bacteria, thus, the term endotoxins is also used interchangeably
but not correct entirely. Both G+ and G- bacteria produce pyrogens, however,
the pyrogens of G-ve bacteria are more potent. The pyrogens are heat stable
up to some extent, thus, withstand normal sterilization temperatures.

Depyrogenation
Depyrogenation is the removal of pyrogen. This is achieved by the following
methods.
Inactivation - Application of very high dry heat (2500P) for not less than 30
minutes is the desired method for rendering material pyrogen free.
Removal of pyrogen by distillation
Detection and quantification of Pyrogens
1) In-vivo pyrogen (rabbit) test
In-vivo pyrogen test involves the evaluation of the presence of pyrogens
in parenteral sample by quantitative fever response produced in rabbits. The
principle is based on the fact that the human and rabbits are equally
responsive to pyrogen injected intravenously on a dose per weight basis.
This test requires the following.
Test animals: healthy adult rabbits (of either sex) weighing not less than
1500 gm (1.5kg). The animals have been properly maintained in terms of
environment and diet prior to the performance of test. The animals are
screened for their temperature.
Their control temperature must not differ more than 1°C from each other.
Any individual animal having temperature 39.8°C or less than 38.0°C is
excluded from the test.
The rabbit-retaining boxes are required to house the rabbits. These boxes
"hold" the rabbits so that the temperature can be noted easily during test.
The specific directions given in the individual monograph must be followed
for the products.
The sample to be tested is injected with a slower rate to the animals. The
dose of the sample if not specified should be smaller than 10 ml/kg. Special
preliminary steps are required and thus, consideration must he given for the
products requiring; 1) dilution, 2) pH adjustment, and 3) isotonicity
adjustment.

PROCEDURE
The control (baseline) temperature of three rabbits is determined. The
sample is injected into the ear vein of each of three rabbits which are held in
the retaining boxes. A dose of 10ml/kg of body weight is used unless
specified in the individual monograph. The temperature of each rabbit is
determined at 1, 2, and 3 hours subsequent to the injection of sample. The
difference between the initial and final temperatures of each rabbits is noted.
Any increase in temperature is taken to be the response of sample injected.

Interpretation of the results

The material under examination meets the requirements for apyrogenicity if
no rabbit shows an individual rise in temperature of 0.6°C or more above its
respective control temperature OR the sum of the temperature rise of 3
rabbits does not exceed 1.4°C. If the results are not within the limits, the
test is repeated for additional 5 rabbits and the result is considered for the
eight rabbits. After repeating, the material under examination meets the
requirements if not more than 3 out of eight rabbits show individual rise in
temperature of 0.6 °C OR the sum of rise in the temperature in eight rabbits
does not exceed 3.7°C.
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.

Advantages of Rabbit Test

The human and rabbits are equally responsive to threshold levels of the
pyrogens.
2) Limulus Amebocyte Lysate 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). 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 and protein 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 buffersolution 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
7. It is more accurate
PYROGEN TEST

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.
PYROGENS:
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.
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 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 or
It has been used at any time in the pyrogen test in which the mean response of the rabbit
in the group exceeds 1.2C.
MATERIALS NEEDED:
(a). THERMOMETERS:
The thermometer or electrical device used should indicate the temperature with a great
sensitivity and should be inserted in the rectum of the rabbit to the depth of about 5.2cm (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.
(b). GLASSWARE, SYRINGES AND NEEDLES:
All the glassware, syringes and needles must be thoroughly washed with water and heated in a
hot air oven at 250C for 30 minutes or at 200C for an hour.
(c). RETAINING BOXES:
The retaining boxes for rabbit in which the temperature is being measured by and 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 of the three days before testing the product, inject pyrogen free isotonic NaCl solution
(10ml/kg body weight warmed at 38.5C 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 0.6C 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 38.5C 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.
The volume of the injection should be not less than 0.5ml/kg body weight and should not be
more than 10ml/kg body weight.
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 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 30 minutes beginning at least 90 minutes
before 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:
It is having an initial temperature higher than 39.8C or lower than 38.0C.
It is showing temperature difference more than 0.2C between two successive readings
taken during the 90 minutes.
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.
Depending on the results obtained tabulate the results in the following manner.

NUMBER MATERIAL PASSED IF MATERIAL FAILED IF

OF SUM OF RESPONSE SUM OF RESPONSE
RABBITS DOES NOT EXCEED. 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
SEALING OF STERILE PREPARATIONS

E) Sealing
The container should be sealed in the aseptic area immediately adjacent to
the filling machine. In addition to retaining the content of the sterile product,
sealing of containers assures sterility of its contents. Temper-proof sealing is
essential so as the sterility can be ensured until usage. Different approaches
have been used for sealing of ampoules and the bottles.

Sealing of ampoules
The ampoules can be sealed either by tip or bead seal or pull seal. Both of
the methods require heating with high-temperature oxygen flame. During
sealing, the heating must be even and carefully controlled to avoid distortion
of the seal. It is sometimes necessary to displace the air in the space within
the ampoule above the product to prevent decomposition. This may be done
by introducing a stream of inert gas, such as nitrogen or carbon dioxide,
during or after filling with the product. Immediately thereafter, the ampoule
is sealed before the gas can diffuse out. The tip seals are made by melting
sufficient glass at the tip of the ampoules neck to form a bead of glass and
close the opening. Thus, tip seal is also known as bead seals since a bead is
formed during melting of the neck. Excessive heat of air and gases in the
neck cause expansion against the soft glass with the formation of fragile
bubbles at the point of seal. Open capillaries at the point of seal or cracks
result in leakers. Fracture of the neck of ampoule often occurs during sealing
if wetting had occurred at the time of filling, Also wet glass at the neck
increases the frequency of bubble formation and contaminating deposits of
carbon or oxides as a result of the effect of the heat of sealing on the droplet
of the product. Pull seals are made by heating the neck of the rotating
ampoule below the tip, then pulling the tip away to form a small, twisted
capillary just prior to being melted closed. Pull sealing is a slower process,
but the seals are more reliable than those from the tip sealing. Powder
ampoules or other types having a wide opening must be sealed by pull-
sealing.
With some sensitive products, it may be necessary to seal the ampoules with
pull-seals to prevent combustion produces of the flame from entering the
ampoule at the time of sealing, as might occur with tip-sealing.

Sealing of bottles, cartridges and vials

The closure is to be slide from a rotating or vibrating drum to the bottom of
a chute, where it is positioned over a container ready for insertion by a
plunger or some other pressure device which is followed by stoperring. To
facilitate sliding of rubber closures, their surface is halogenated or coated
with silicon which reduces the friction during slipping into the container’s
mouth.
Aluminum caps are used to hold rubber closures in place. Single caps may
have a permanent center hole or a center that is torn away at the time of
use to expose the rubber closure. When applied, the bottom edge of the
aluminium cap is bent (crimped) around and under the lip of the glass
container. It cannot be removed without destroying the cap but perforation
permit tearing away the portions of the cap to be discarded. Crimping can be
achieved using the heavy duty crimping machines.

FILLING OF POWDERS

Sterile solids are more difficult to subdivide accurately and precisely into
individual dose containers than are liquids. The rate of flow of the solid
materials tends to be slow and irregular, particularly if the powder is finally
divided. Small granular particles flow most evenly. Uniform particle size and
good flow properties of solids are necessary for uniform and effective filling
by machines. For powder showing poor flow, the containers with a relatively
large opening must be used, even so, the filling rate is slow and the risk of
the spillage is ever present. For these reasons, the tolerances permitted for
the contents of such containers must be relatively large.
Relatively freely flowing solids are filled using filling machines. One type of
machine for delivery of measured quantities of solid material employs an
augar in the stem of the funnel-shaped hopper. The size and rotation of the
augar can be adjusted to deliver a regulated volume of granular material
from the funnel stem into the container.
In another filling machine, a adjustable cavity in the rim of the filling wheel
is filled by vacuum as the wheel passes under the hopper. The contents are
held by vacuum until the cavity is inserted over the container when a jet of
sterile air discharges the solids. This machine also dispenses dry solid that
flow less freely.

D) FILLING PROCEDURE

D) Filling procedure
The filling process has been categorized as the filling of low density and
viscosity liquids, filling of viscous liquids and filling of solids. Filling
equipment has a reservoir to hold bulk product. The reservoir is connected
to delivery tube to dispense product into container. A mean is provided for
repetitively forcing a measured volume/amount through the orifice of a
delivery tube. The accuracy and the precision of the machine filling of sterile
liquids vary with the method. Therefore, a method is selected to provide the
degree of accuracy and precision required by the nature of the product. The
slightest excess is required in each container to provide for the loss that
occurs at the time of the withdrawal of dose at the time of administration
due to adherence of container content to the wall of container and retention
in the syringe. Filling machines should be designed so that the part through
which the liquid flows can be easily demounted for cleaning and for
sterilization. These parts also should be constructed of non-reactive
materials, such as borosilicate glass or stainless steel. Syringes are usually
made of stainless steel, when the pressure required for delivery of the
viscous liquid or large volumes would be useful for glass syringes.

Filling of low viscosity small volume liquid preparations

A liquid may be subdivided from a bulk container to individual dose
containers more easily and uniformly than a solid. Mechanical subdivision of
a mobile, low density liquid can be achieved with light-duty machinery.
Certain fundamental features are found on all filling equipments for liquid
preparations. The filling equipment has a reservoir to hold the bulk of the
liquid preparation to be filled into containers. A means is provided for
repetitively forcing a precisely measured volume of the liquid through the
orifice of a delivery tube designed to enter the constricted opening of a
container. The size of the delivery tube is governed by the opening in the
container to be used, viscosity and density of the liquid and the speed of the
delivery desired. The tube must enter freely into the neck of the container
and deliver the liquid deep enough to permit air to escape without sweeping
the entering liquid into the neck or out of the container. To reduce the
resistance to the follow of the liquid, the tube should have the maximum
possible diameter. Excessive force of delivery causes splashing of the liquid
and troublesome foaming, if the liquid has a low surface tension. The
delivery of relatively small volumes of liquids is usually obtained with
pressure obtained from the strokes of the plunger of a syringe. The stroke of
the syringe forces the liquid through a two-way valve that provides for an
alternate filling of the syringe from a reservoir and delivery to a container. A
drop of liquid normally hangs at the tip of the tube after a delivery. When
the container to be filed is an ampoule, withdrawal of the tube without
wetting the long restricted neck is almost impossible, unless the hanging
drop of the liquid is retracted. Thus, a retracting device is designated as a
part of the most filling machines.

Filling of low viscosity large volume liquid preparations

Sterile solutions of relatively low potency dispensed in large volumes (up to
1 liter) do not normally require the precision of filling that is required for
small volumes of potent injectables. Therefore, the liquid is filled into the
bottles by gravity, pressure or vacuum filling devices. Generally, gravity
filling is relatively slow, but is accomplished with simpler means. A liquid
reservoir is positioned above the filling line, with a hose connection from the
reservoir to a shut-off device at the filling line. The shut-off device is usually
hand operated, and the bottles are filled to graduations on the bottles.
The pressure pump filler often is operated semi-automatically and differs
from the gravity fillers, principally in that the liquid is under pressure. It is
usually equipped with the overflow tube connected to a receiver to prevent
excess flow of the container. Vacuum filling is commonly used in faster filling
lines for large liquid volumes because it is more acceptable for automation. A
vacuum is produced in a bottle when a nozzle gasket makes a seal against
the tip of the bottle to be filled. The vacuum draws the liquid from a
reservoir through the delivery tube into the bottle. When the liquid level
reaches to a level of an adjustable overflow tube, the seal is mechanically
loosened and the vacuum is released. Any liquid that had been drawn into
the vacuum line is collected in a trap receiver and then returned to the
reservoir.

Filling of high viscosity liquid preparations

The viscous, sticky or high density liquids require much more heavy
machines to withstand the pressure required to dispense them in individual
containers. Thus, compared to the plunger-syringe assembly for filling of low
viscosity liquid, for heavy, viscous liquids, a sliding piston valve provides
more positive action. Emulsion, suspensions and semisolid preparations
often require specially designed filling equipments because of their high
viscosity. To obtain a reasonable flow rate of the emulsion and suspension,
high pressure must be applied or container with large openings must be
used to permit the entry of large delivery tubes. Sometimes the jacketed
tanks can be used to raise the temperature of the product to facilitate filling
by lowering its viscosity. It is normally necessary to keep suspensions and
sometimes emulsion, constantly agitated in the reservoir during filling so
that the product remains homogeneous and each subdivided unit contains
the required amount of drug.

C) FORMULATION OF STERILE PRODUCT

C) FORMULATION OF STERILE PRODUCT

The product formulation is sometimes, just compounding of the ingredients.
In other situations, the parenteral products are formulated as emulsions,
suspensions, cream and ointments. All the process are undertaken in strict
aseptic conditions.

Compounding of ingredients
The ingredients should be compounded under clean environmental
conditions. A sterile condition is usually not required since it may not be
possible or feasible to sterilize some of the ingredients or equipment, e.g.,
large tanks. Whenever possible, however, the equipments and the
ingredients should be sterile to reduce the microbial load.
The compounding process should meet the rigid standards accepted in
pharmaceutical procedures, regardless of the batch size, recognizing that
small multiple errors may be additive. In large batches particular attention
must be given to achieving and maintaining homogeneity of solution,
suspensions and mixtures, maintaining a given temperature and accelerating
cooling. The order of mixing ingredients may become highly significant, for
example, owing to the physical problem of disturbing a pH adjusting
ingredient throughout a large tank of liquid. Compounding problems for
large batches of product often are different from those of the small batches.

B) Sterilization methods

Six sterilization methods are available and are selected based on the item,
material of product to be sterilized. These include; 1) sterilization by steam,
2) sterilization by dry heat, 3) sterilization by ethylene oxide, 4) sterilization
by filtration, 5) Lyophilization, and 6) sterilization by -radiations. A detail
description of the methods has already been given in previous classes.

CLEANING RUBBERY PLASTIC COMPONENTS

The rubber closures are usually washed by mechanical agitation in a tank of

hot detergent solution (such as 0.5% sodium pyrophosphate) followed by a
series of through water rinses, the final rinse being WFI. The objective is to
remove the surface debris accumulated from the molding operation and from
handling and leachable constituents at or near the surface. Part of the debris
is attached and held on the surface by electrostatic forces. Similarly, plastic
materials accumulate surface debris.
The multiple objectives for washing closures and other parts include
loosening debris, minimizing abrasion and sweeping away the loosened
debris.

CLEANING OF CONTAINERS, GLASSWARE AND METAL WARES

Like instruments, the unused containers are contaminated with dust, fibers,
and chemical films. These are removed by vigorous treatment with hot
detergents. The containers are inverted on spindles in the front of the
cleaning machine and are automatically conveyed in an inverted position.
During rotation of the cleaning machine, they are carried through a series of
rigorous, high pressure treatment including hot detergent, hot tap water and
final rinses with distilled water. Because many containers have restricted
openings, it is essential that the treatments in any washer be introduced
though tubes into each container with smooth outflow. For ampoules or
containers with a markedly constricted opening that makes water drainage
incomplete, the final treatment is usually a blast of clean air to blow out
remaining water.

After cleaning, it is essential that the clean containers be protected from

dust and other particulates that might be present in the environment.
Therefore, the clean containers are often removed from the rinser and
placed in clean stainless steel boxes for sterilization under the protection of
HEPA filtered airflow. Glassware and metal ware (small) may also be
contaminated from previous use and can also be mechanically like
containers in their converted position during automatically conveying
through a series of rigorous, high pressure treatment including hot
detergent, hot tap water and final rinses with distilled water. In cleaning new
glassware, the detergent treatment is usually eliminated, and with it the risk
of the detergent residue.
With rinsing alternatively by hot (preferably clean steam) and cold
treatments should be used to lessen the debris. Final rinses should be done
with filtered WFI.

A) CLEANING OF EQUIPMENTS, CONTAINERS AND GLASSWARE (1)

Cleaning of Equipments
The equipments to be used in the processing of the sterile products must be
thoroughly cleaned. The new and unused equipments are contaminated
principally with dust, fibers, and chemical films which usually are relatively
easy to remove often by rinsing only.
Debris that is more dangerous and more difficult to remove may be present
as residue form the previous use. These are removed by vigorous treatment
with hot detergents.
Whenever possible, large equipments should be disassembled so that each
part can be thoroughly scrubbed and clean with particular attention given to
screw threads, joints and other dirt collecting structures. After cleaning, the
equipments should be rinsed several times with final rinse with water for
injection (WFI). Just prior to re-use, large cleaned tanks and similar
equipments should be rinsed thoroughly with WFI. Reserving of the
equipment for use with only one type of the product reduces the cleaning
problems.

A new method for large tanks, pipe lines and associated equipments that can
be isolated and contained within a process unit has been developed and
identified as clean in place
(CIP) system. Under this system, cleaning of the dedicated instruments for
specific products is accomplished primarily with high pressure rinsing
treatments, delivered automatically within the equipment. This is usually
followed by steam sanitization through the same system.

STERILE PRODUCTION PROCESS

The general production process started from the accumulation of raw

materials, ingredients and packaging components and then combining of the
ingredients of the formula into a product. The prepared product is enclosed
in the individual containers for distribution. Sterilization during production
and/or after production is also employed.
During production, in-process quality control and the finished product quality
control is also performed. The required equipments are cleaned prior to the
start of the sterile production process.

STANDARD OPERATING PROCEDURES

To enhance the assurance of the successful manufacturing operations, all

process steps must be carefully written. The written process steps are often
called standard operating procedures (SOP). No extemporaneous changes
are permitted in the SOP. Any change in SOP must go through the same
approval steps as the original written SOP. According to the FDA guidelines
on the good manufacturing practices, extensive records must be kept to
assure, at the end of the production process in that all steps have been
performed as prescribed.

Such in-process control is essential to assure the quality of the product.

Since this assurance is even more significant than those from product
release testing. Production of quality product is a result of continuous,
dedicated efforts of the quality assurance, production and quality control
personnel within the plan in developing, performing and confirming effective
SOP.

The process equipments and the components of the containers, cleaned

thoroughly according to the required specifications are assembled in a clean
environment. The assembled components are preferably sterilized and
depyrogenated prior to use. All equipments and the supplies, introduced into
the aseptic filling areas should be sterilized.
The outer surfaces of the boxes, packages, or equipments should be wiped
with a disinfectant solution as they are transferred to clean room. All the
supplies must be introduced into the aseptic filling room in such a manner
that the aseptic state of these room is maintained, thereby, preventing the
introduction of environmental contamination into the product while it is
being subdivided into individual containers. After sealing of containers,
contamination cannot enter into the container and the product. Thus the
product is sealed in its final container within the aseptic room from where it
is transported to packaging area. This area is maintained clean but need not
meet the standard imposed for the aseptic room or for the sterile
compounding room. Packaged products are placed in quarantine storage
until all tests have been completed and in-process control records have been
evaluated.

PERSONNEL FOR STERILE PREPARATIONS

F) Personnel
The most ideally planned processes can be rendered ineffective by personnel
who do not have the right attitudes or training. The personnel who produce
sterile products usually are non-professional person, supervised by those
with professional training. To be effective operators, they must inherently
neat, orderly, reliable and alert and have good manual dexterity. They
should be appreciative of the vital role that every movement lies in
determining the quality of final product, it its freedom from contaminants.
All employees should be in good health and should be subjected to periodic
physical examinations. They should understand their responsibility to report
the developing symptoms of cold, sore throat or other infectious diseases, so
that they can be assigned to
a less critical area until they have fully recovered.
The attire won by personnel in the aseptic areas usually consists of sterile
coveralls, hoods, face masks and show covers. Sterile rubber gloves also
may be required.
Personnel entering the aseptic areas should be required to follow a definite
preparatory procedure. This should include removing at least outside street
clothing, washing the hands and arms thoroughly with a disinfectant soap,
and donning the prescribed uniform.
A full body water and soap shower would be essential n most biologic
products processing plants – usually, both when entering and leaving the
area to control contaminations in both directions, between personnel and the
product. Since people are continually shedding viable and non-viable
particulate matter from body surfaces, uniform are worn to help to control
this emission. The uniform should, preferably be of coverall type and made
of synthetic fibers such as Dacron. Dacron cloth
is made of a continuous fiber, which makes it essentially lint-free and in air
conditioned room, is acceptably comfortable.
ANOTHER SYSTEM OF CLASSIFICATION FOR CLEAN ARE

ANOTHER SYSTEM OF CLASSIFICATION FOR CLEAN AREA

Number of Particle Diameter (um)

Class
0.1 0.3 0.5 5
1 35 3 1
10 350 35 10
100 300 100
1000 1000 7
10,000 10,000 70
100,000 100,000 700

Federal Standard 209 (FED STD 209) For Clean Area

1. Class 100,000: Particle count not to exceed a total of 100,000 particles

per cubic foot of a size 0.5μ (micron) and larger or 700 particles per cubic
foot of a size 5.0μ (micron) and larger.
2. Class 10.000: Particle count not to exceed a total of 10,000 particles per
cubic foot of a size 0.5μ (micron) and larger or 65 particles per cubic foot of
a size 5.0μ (micron) and larger.
3. Class 1,000: Particle count not to exceed a total of 1000 particles per
cubic foot of a size 0.5μ (micron) and larger or 10 particles per cubic foot of
a size 5.0μ (micron) and larger.
4. Class 100: Particle count not to exceed a total of 100 particles per cubic
foot of a size
0.5μ (micron) and larger.
(The Clean Room class is generally achieved in the "at rest" state when
there are no people in the room)

British Standard 5295 For Sterile Area

British Standard 5295

Class 1: The particle counts shall not exceed a total of 3000 particles/m3 of
a size of 0.5μ (micron) or greater. The greatest particle present in any
sample shall not exceed 5μ (micron).
Class 2: The particle count shall not exceed a total of 300,000 particles/ m3
of a size 0.5μ (micron) or greater: 2000 particles/m3 of a size 5μ (micron)
or greater: 30 particles of a size 10μ (micron) or greater.
Class 3: The particle count shall not exceed 1,000,000 particles of a size of
1 micron or greater: 20,000 particles/m3 of a size 5μ (micron) or greater:
4000 particles/m3 of a size 10μ (micron) or greater; 300 particles/m3 of a
size 25μ (micron) or greater.
Class 4: The particle count shall not exceed a total of 200,000 particles/m3
of a size 5μ (micron) or greater: 40,000 particles/m3 of a size 10μ (micron)
or greater: 4000 particles/m3 of a size 25μ (micron) or greater. (3000
particles/m3 at 0.5um converts to about 85 particles/Ft3)

PRODUCTION FACILITIES FOR STERILE PRODUCTS (Continued...)

A) Environmental control
Effective environmental control, both physical and biologic is essential but the level achievable
is related to the characteristics of the facility. Further rigid standards from plant to plant and from
geographic location to another are not appropriate. Allowance also must be made for variation in
control associated with the seasonal conditions.
The standards of environmental control vary depending on the area involved (cleanup,
packaging, compounding or filling) and type of product being prepared. Unquestionably, the
entire area used for the preparation of a product prepared aseptically (without terminal
sterilization) must be maintained under the most rigid control that the existing technology
permits. If the product is to be terminally sterilized, somewhat less rigid biologic control of the
compounding and filling areas may be acceptable. However, rigid standard of cleanliness must
be maintained. High standards of cleanliness, excluding daily use of disinfecting procedures are
usually acceptable for the cleanup and packaging areas.

B) Traffic control
Carefully designed arrangement to control and minimize traffic, particularly ‘in’ and ‘out’ of the
aseptic areas is essential. Access by personnel to the aseptic corridor and aseptic compound and
filling rooms will be only possible through an airlock. Pass-through openings and double ended
sterilizers are provided to permit controlled passage of supplies from non-aseptic to aseptic area.
Persons should be permitted to enter aseptic areas only after following rigidly prescribed
procedures for removing street clothing, washing their hands and putting on gowns, hats, shoes,
facemasks, gloves and other prescribed attire. Once they have entered the aseptic area, they
should not be permitted to move in and out of the area with out regowning.
Personnel assigned to cleaning and packaging should be restricted to these areas.
Unauthorized personnel should never be permitted to enter the aseptic area.

C) House keeping
All equipment and the surrounding work area must be cleaned thoroughly at the end of the
working day. No contaminating residues from the concluded process may remain.
The ceiling, walls, and other structural surfaces must be cleaned with a frequency which is most
appropriate. All cleaning equipments should be selected for its effectiveness and freedom from
lint-producing tendencies. It should be reserved for use in the aseptic areas only.

D) Surface disinfection
After through cleaning all surfaces should be disinfected, at least in the aseptic areas. An
effective liquid disinfectant should be sprayed or wiped on all surfaces. Irradiation from
ultraviolet lamps that are located provide adequate radiation intensity on the maximum extent of
surfaces in a room and that are maintained free from dust and films further reduces the viable
microorganisms present on the surface and in the air.
Ultraviolet rays may be particularly useful to irradiate the inside exposed surfaces of the
processing tanks, surfaces under hoods. The surface of the conveyor belts and the similar
confined surfaces those are otherwise, difficult to render aseptic. However they cannot reach
unexposed surfaces such as pipe connections to tanks, the undersides of conveyors and the inside
of containers.
The UV lamps must be kept clean and care must be taken to check for a decrease in effective
emission, a natural occurrence due to a change in the glass structure with aging.
E) Air control
In any area occupied by personnel, air must be exchanged at frequent intervals. Fresh outside or
recycled air must first be filtered to remove gross particulate matter. A spun glass, cloth, or
shredded polyethylene filter may be used for this preliminary cleaning operation.
At times, more than one pre-filter may be used in series, the first one is quite large and the next
somewhat smaller pore size to provide a gradation of particle size removal from heavily
contaminated air. To remove finer debris down to the submicron range, including
microorganism, a high efficiency particulate air (HEPA) filter is used. The HEPA filter has been
defined as at least 99.97% efficient in removing particles of 0.3um size and larger and composed
of glass fibers and filters or electrostatic precipitators may be employed. Air passing though
these units can be considered virtually free from foreign matter. Another air cleaning system
washes the air with a disinfectant and controls the humidity at the same time.
Blowers should be installed in the air ventilation system upstream to the filters, so that all the dirt
producing devices are ahead of the filter. The clean air is normally distributed to the regulated
areas by means of metal (preferably stainless steel) ducts. Since it is practically impossible to
keep these ducts as clean as required, it is normally preferred to install HEPA filters at the where
the clean air enters the controlled room. Alternatively, the ducts may be replaced with a room (a
plenum) usually above the production area, into which clean air is blown and then distributed
through opening into each of the process rooms. The entire plenum can be kept clean and aseptic.
The clean and aseptic air is distributed in such a manner that it flows into the maximum security
room at the greatest volume flow rate, thereby producing a positive pressure in these areas. This
prevents unclean air from rushing into the aseptic area though cracks, temporarily opened doors
or other openings. The pressure is reduced successively so that the air follows from the
maximum security area to other less critical areas for return to the filtration system. At the intake
end of the system, fresh air usually about 2% is continually introduced for the comfort and needs
of the personnel. Further, the air is usually conditioned with respect to the temperature and
humidity for the comfort of the personnel and sometimes to meet the special requirements of a
product.
Horizontal laminar flow hood Vertical laminar flow hood
A relatively new air control system, based on laminar flow principle, has greatly improved the
potential for environmental control of aseptic areas. Currently, it is the only means available for
achieving a class 100 clean room. A class 100 clean room is defined as a room in which the
particle count in the air is not more than 100 per cubic feet of 0.5um and lager in size. The air
filtered through HEPA filter is blown evenly out of the entire back or top of the work bench or
entire side or from ceiling of a room. The air flow must be uniform in velocity and direction
throughout any given cross-section of the area, being exhausted from the opposite side. The air
velocity employed should be 100 k 20 ft/min.
Contamination is controlled because it is swept away with the airflow.
Although class 100 work environments are normally specified for the most critical aseptic and or
clean operations associated with the parenteral preparations, achieving such levels of cleanliness
is expensive and requires effective maintenance and monitoring. It should be recognized that not
all operations associates with parenteral medication require such an environment. To such an end
other classes are defined. For example, a class 10,000 room is one in which the particle count is
not more than 10,000 per cubic feet of 0.5 um and large size. Such a cleanliness level is usually
considered suitable for buffer areas around class 100 worksites in which operations such as
handling of pre-cleaned containers, process filtration and aseptic gowning of personnel may be
performed. Still less stringent requirements would be applied to laboratories, stock staging areas,
and finish packaging where a class 100,000 or similar cleanliness levels would be considered
suitable.
Different classes and standards of clean rooms:
The determination of how clean an area is, depends on the classification that it has been designed
with different standards. Four different classes are described according to British Standard
system 5295 and Federal Standard 209 (FED STD 209).