This document provides information on process validation for ampoules and vials. It discusses the key steps in prospective, concurrent, and retrospective validation including pre-validation requirements. Critical process parameters for vial washing, depyrogenation, filling, sealing, and other sterile processing steps are identified. Process validation aims to demonstrate the manufacturing process will consistently produce sterile products meeting specifications. Quality control checks like leak testing, pyrogen testing, sterility testing, and particulate evaluation are summarized.

1. PROCESS VALIDATION OF AMPOULES AND VIALS
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PRESENTED BY
Khushboo Pasbola
M.pharm(QAT)
Dept. of Pharmaceutics
SGRRITS,Dehradun
FACILITATED BY:
Dr. Alka N.Chaudhary
Professor
Dept. of Pharmaceutics
SGRRITS, Dehradun

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Sterile Liquid Products
(PARENTERALS)
AMPOULES AND VIALS
PROCESS VALIDATION

3. Process Validation
‰Process validation is establishing
documented evidence which demonstrate
that the manufacturing process will
consistently produce a product meeting its
predetermined specifications and quality
Characteristics.
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4. Process Justification:
 ‰To identify critical process steps &process parameter
of Mixing process
 ‰To determine the suitable Hold time Period ‰
 To confirm the analytical tests that will have to be
performed ‰to define the optimal parameters
throughout the overall ampoule filling process to
consistently produce the finished products(filled
ampoules)which meet the established specifications. ‰
 To assure that the product is sterile after sterilization
process
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5. 1. Prospective validation
 Prospective validation is establishing documented evidence
prior to process implementation that a system does what it
proposed to do based on preplanned protocols.
 It is conducted prior to the distribution of either a new
product, or product made under a revised manufacturing
process, where the revisions may affect the product’s
characteristics.
 In prospective validation process validation, validation
protocol is executed before process is put into commercial
use (i.e. qualification trails). This type of process validation
is usually carried out in connection with introduction of
new drugs.
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Type of Process Validation

6. 2. Concurrent validation
This validation involves in process monitoring of critical
processing steps and product testing.
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3. Retrospective validation
Retrospective validation establishes documented evidence
that a system does what it is supposed to do based on a
review and analysis of historic information. It is establishing
document conducted for a product already marketed based on
extensive data accumulated over several batches and over
time. It is normally conducted on a product already being
commercially distributed and it is based on accumulated
production, testing and control data.

7. 4. Revalidation
 It is the repetition of a validation process or a specific
part of it. This is carried out when there is any change
or replacement in formulation, equipment, plant or
site location, batch size and in the case of sequential
batches that do not meet product and process
specification.
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8. Pre-validation Requirements
 ‰Preventive Maintenance for Facilities and Utilities
 ‰Calibration of Equipment
 ‰Cleaning Validation
 ‰Equipment &System Qualification
 ‰Raw Materials/Components/Test Methods
 ‰Process Justification
 ‰Change Control
 ‰Training operators
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9. Vial washing and depyrogenation
 Water and compressed air pressure
The vials are washed first by series of water at a high
pressure. The pressure of the water should be
maintained throughout the process as the pressure is
directly proportional to the effectiveness of the
washing process. If the pressure is less than the
acceptable value it may lead to improper washing.
Compressed air is used for drying the washed vials
before it reaches the tunnel. Thus any deviations in the
pressure maintained will leads to improper drying
which will affect the depyrogenation of the vials.
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10. • Clarity check
The washed vials are checked manually for the effectiveness
of the washing process. The vials which are broken or
containing any dirt can be identified during these check’s
which prevent the rejections after filling.
 Depyrogenation temperature and conveyor belt
speed Vial depyrogenation is another critical factor to be
checked. Depyrogenation of vials can be achieved at a temperature
between 280˚C to 350˚C. If the temperature is not maintained
throughout the process it may affect the depyrogenation of the
vials. The tunnel should be qualified before starting the process.
During the qualification stage the depyrogenation temperature
and the conveyor belt speed for the different vial size are done and
established. The validated limits should be followed during the
process.
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11. •∆P (across the HEPA filter and across the zones &
rooms)
Pressure differentials in the tunnel is important to
ascertain that the correct degree of over pressure is
maintained relative to the adjacent areas of lower
classifications in order to minimize the contamination
drawn into the controlled environment from its
surroundings. The ∆P should be maintained within
limits to maintain a controlled environment. The
difference in ∆P may be due to blocked or partially
blocked HEPA filters. The ∆P across various zones of
tunnel and the ∆P between the tunnel cooling area and
vial receiving area should be maintained.
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12. Vial filling and sealing
 1 .Gowning procedure
The persons entering the sterile area should not
contaminate the area by shedding contaminants from own
body. The person should follow the gowning procedure
strictly. The gowning qualification is done by taking swabs
from the gowned persons at the commonly used parts of
the body on consecutive three days and incubated for
checking the presence of any viable organism in it. The
results showing less than the alert level is the criteria for
acceptance. Person without proper training may lead to
product contamination, thus proper training is given to all
personnel entering sterile area.
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13. 2.Filling speed and fill volume
 The filling speed of the machine should be validated
during the machine qualification stage. The filling
speed depends upon the size of the vial and volume of
the liquid filled. The fill volume may be altered due to
increasing or decreasing the speed of the machine and
also depends on the product physical nature. The
changes from the established limits may leads to
reduced extractable volume. Thus it should be
frequently monitored by doing fill volume checks by
using calibrated syringes or measuring cylinders.
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14. 3. Filter integrity testing
 Fiteration is the only process of sterilization in
aseptically filled products, thus to confirm the
sterilization has achieved filter integrity should be
done. The integrity of the sterilization filter should be
verified before use and should be confirmed
immediately after use by an appropriate method such
as a bubble point, or diffusive flow or pressure hold
test.
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15. 4. Sealing of vials
 The vials filled asepticaly should be closed and
sealed immediately after filling of the solution.
The sealing gives the proper closing of the vials.
The sealing activity can be confirmed by doing
leak testing for the sealed vials frequently. Sealing
prevents the leakage of the containers during the
transporting or shipping.
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16. Objective of process validation for sterile product:-
 To build sterility into a product
 To demonstrate to a certain maximum level of probability that the
processing & sterilization methods have established sterility to all units
of a product batch.
 To provide greater assurance & support of the results of the end-
product sterility test.
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17. Terms to be considered for
validation of aseptic process :
 Aseptic filling: Operation whereby the product is
sterilised separately, then filled and packaged using
sterilised containers and closures in critical processing
zones.
 Bio burden: Total number of viable microorganisms
on or in pharmaceutical product prior to sterilisation.
 High efficiency particulate air (HEPA) filter:
Retentive matrix designed to remove a defined
percentage of particulate matter of a defined size.
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18.  Aseptic Operation: Operation whereby the product is
sterilized separately by filtering through 0.2 µor less
filter, then filled and packaged using sterilized
containers and closures in critical processing zones
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HEPA FILTERS

19.  HVAC: Heating, Ventilation and Air Conditioning
 Integrity test: Test to determine the functional performance of
a filter system
 Media fills: Method of evaluating an aseptic process using a
microbial growth medium. (Media fills are understood to be
synonymous to simulated product fills, broth trials, broth fills
etc.).
 Sterile: Free of any viable organisms.
(In practice, no such absolute statement regarding the absence
of microorganisms can be proven)
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20.  Sterilisation: Validated process use to render a product free of
viable organisms. (Note: In a sterilisation process, the nature of
microbiological death is described by an exponential function.)
 Sterility assurance level (SAL): Probability that a batch of
product is sterile.
 Sterility test: Test performed to determine if viable
microorganisms are present.
 Vent filter: Non-shedding porous material capable of removing
viable and non-viable particles from gases passing in and out of
a closed vessel.
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21. Sterility Test :-
 The sterility test can provide useful information on the
validation status of aseptic process.
 However;
The sterility test applied to the finished product should only be
regarded as the last in a series of control measures by which
sterility is assured. The test should be validated for the
product(s) concerned.
 It is important to compare the retest rate for aseptically
processed products against that for terminally sterilised
products. If aseptically processed products have a higher rate
then this may be indicative of sterility problems not identified
during validation.
 This is not an unusual situation as validation cannot take into
account all the possible permutations and combinations in
equipment, personnel and processes 21

22. Quality Control Checks for Sterile products
a) Conductivity measurement
b) Volume filled
c) Temperature for heat sterilized product
d) Environmental control tests
e) Visual inspection
a) Leaker Test
b) Pyrogen Test
c) Particulate Test
d) Sterility Test
e) Uniformity of content
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23. Leaker Test
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PROCEDURE
• Leakers are detected by this
process in a visible manner.
• Ampoules are placed in a vacuum
chamber.
• Completely submerged in a deeply
colored dye solution of about 0.5 to
1% methylene blue.
• A negative pressure is applied
within the ampoule. Subsequent
atmospheric pressure causes the
dye to penetrate an opening thus
making it visible after the ampoule
has been washed.

24. Pyrogen Test
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1. LAL test 2. Rabbit test

25. LAL Bacterial Endotoxin Test
 The LAL (limulus amebocyte lysate) Assay is an in
vitro assay used to detect the presence and concentration of
bacterial endotoxins in drugs and biological products.
 Endotoxins, which are a type of pyrogen, are
lipopolysaccharides present in the cell walls of gram-negative
bacteria.
 Pyrogens as a class are fever-inducing substances that can be
harmful or even fatal if administered to humans above certain
concentrations.
 Water can be a source of pyrogens, so it may be important to
routinely monitor water systems using the bacterial
endotoxins test.
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26.  The solution of endotoxins containing preparation is added to
the lysate derived from heamolymph cells of horseshoe crab
(limulus polymhemus).
 The result of the reaction is turbidity or precipitation or
gelation of the mixture.
 This is used as a quantitative measure to estimate the endotoxin
content.
 The rate of reaction depends upon conc. of endotoxins, pH,
temperature and presence of clotting enzyme system and
clottable proteins from lysate.
 The quantities of endotoxins are expressed in defined
Endotoxin Units (EU). Also 1 EU is equal to 1 IU.
 The endotoxin limit for a given test preparation is calculated
from the expression K/M; where M is maximum dose
administered to adult per kg per hour.
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27. Pyrogen Test
Fever response of rabbit
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• Withheld food in the day of
experiment.
• Record the initial
temperature of the rabbits,
any rabbit show temp. more
than 39 ˚C, should be
excluded.
• Inject the sample into the ear
vein of each rabbit.
• Check the temperature
after 30 minutes, 1, 2 and 3
hours.

28. Sterility Test
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a. Membrane filteration
Method
 Media suitable for membrane
filteration method are:
i. Fluid thioglycolate medium
ii. Soya-bean casein digest medium
• Wash the filters with fluids to
remove inhibitory properties,
cutting the membranes
aseptically into equal parts and
transferring one of the parts to
each type of culture medium
used.
• The media are then incubated
under prescribed conditions (30-
35˚C / 20-25˚C for NLT 7 days)
• Observe the growth and interpret
the results.

29. Direct Inoculation Method
Parenteral preparation
Culture medium
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• This method is used
when membrane
filtration is not possible.
The sample is inoculated
directly into the media.
Result:
• If no microbial growth
in the media then the
sample passes test for
sterility.

30. Particulate Evaluation
 It has been shown that particles of lint, rubber, insoluble
chemicals, and other foreign matter can produce emboli in the
vital organs of animals and human beings.
 The USP specifies that good manufacturing practice (GMP)
requires that each final container of an injection be subjected
individually to a visual inspection and that containers in which
visible particles can be seen should be discarded.
 Therefore, all of the product units from a production line
currently are being inspected individually by human inspectors
under a good light, baffled against reflection into the eye and
against a black-and-white background.
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31. References:
 PROCESS VALIDATION OF PARENTERAL
FORMULATION Sudarshan Kakad1*, Kiran Wale1,
Mahesh Balsane1, Dr.K.S.Salunkhe1, Dr.S.R Chaudhari
World Journal of Pharmaceutical Research
 Google search engine
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