validation of sterile product

1. Validation of sterile product
Guided by-
Dr Tejal Mehta
HOD of Pharmaceutics
Institute Of Pharmacy
Nirma University Submitted by-
Akansha Sharma
14mph101
Department of pharmaceutics

2. Introduction
• Sterile products are the unique dosage forms that are
free from microorganisms, freedom from pyrogens,
freedom from particulates.
• They have high standard of purity and quality.
• The ultimate goal is absence of microbial
contamination.
• Because of these features there are differences in the
design and specifications of premises, equipment, and
processes of sterile products.

3. Principle involve in validation of sterile product
• To build sterility into a product.
• To demonstrate to a certain maximum level of
probability that the processing
and sterilization methods have established sterility to
all units of a product batch.
• To provide greater assurance and support to all the
results of the end products sterility test

4. Process Of Microbial Destruction
• Regardless of type of lethality induced by
sterilization process whether it be heat,
chemical ,radiation micro-organism will die
according to a logarithmic relationship between
the concentration or population of living cells
and the time exposure or radiation dose to the
treatment.

5. D –value principle and logic
• The D value is a single quantitative expression of
the rate of killing of micro-organism.
• D value = Exposition time required (in minutes) to
kill 1 log (90%) of the micro organisms.
• A D value of 6 means that it takes 6 minutes to
reduce par a factor of 10 (90%) or 1 log the number
of micro organisms.

6. Z -value
• Z value = the number of degrees of temperature
required to obtain a variation of 1 log of the D value.
• Z= T2 -T1
• log D1 - log D2
• If the temperature is lowered, bugs are killed more
slowly and the D value increases (because it takes
more time to kill) .
• Conversely if the temperature is higher, faster the
bugs die, and lower is the D value

7. Continue..
• The Z value is the necessary component of the calculation
which allows the comparison of the spore lethality at
different temperatures.
• Generally accepted Z values assumptions are:
• Steam sterilization Z=10 C
• Dry heat sterilization Z=20 C
• The Z value can be determined by one of the two methods
• -By carrying out experiments at different temperatures to
determine the thermal death times.
• -By carrying out experiments to determine D values at
different temperatures

8. F value
• If the D value is measured at different temperature and
pressure it can be seen that a D value varies with the
pressure. More the D value decreases with a specific
increase of pressure, more powerful is considered the
process.
• F value = a measure of the capacity to inactivate
bacteria in function of the temperature.
• Mathematically the F value is expressed by the rate of
mortality per minute in function of temperature for a
given pressure.

9. Fo Principle
• The Fo value is mentioned both in the USP and in
current good manufacturing practice for large
volume parentral.
Concept of Fo
▫ Lethality factor equivalent to time at 121.1°C
 1 minute at 121.1°C is equivalent to Fo of 1.
 Lethality can accumulate during heat up and cool
down phases.
Typical temperature profile of a heat sterilization
process

10. • Fo is calculated using the following equation:
• Fo = Δt * 10(T-121.1/Z)
• where:
• “Δt” is the time interval between measurements of
temperature (T)
• “T” is the temperature of sterilized product at time (t)
• “Z” is a temperature coefficient which measures the
number of degrees required to change the D-value of
an organism by 1 log

11. • When heat labile products will not withstand excessive
heat treatment, "D121" value studies of product isolates
are necessary to determine the minimum Lethality Factor
(F0) that will provide an acceptable assurance of
sterilization.
• The minimum "F0" value required by a process can be
related to the "D" value of the bioburden by the following
equation:
F0 = D121 (log A - log B)
where:
• "D121" is equal to the time required at 121oC to reduce the
population of the most heat resistant organism in the unit
by 90%;

12. Continue..
• "A" is the microbial count per container; and
• "B" is the maximum acceptable probability of survival ( 1
x 10-6 for pharmaceutical dosage forms).
• F0 values may be solved using the biological approach.
• F0 = D121(log A − log B)
• For example, if D121 = 1.0 min, A = 106, and B = 10−6, F0
is calculated to be
• F0 = 1(log 106 − log 10−6) = 12 min
• Thus, the cycle must be adjusted so that the F0 value
calculated by physical methods (time and temperature
data) will be at least 12 min.

13. Basic Step in validation of sterile product
• Five basic steps are necessary to validate any process
1. Written documentation
2. Manufacturing parameters
3. Testing parameters
4. In-process controls
5. Final product testing

14. Continue..
• Select or define the desired attributes of the product
. example: The product will be sterile.
• Determine the specifications for desired attributes.
• Select the appropriate process and equipments
• Develop and conduct tests that evaluate and
monitor the processes.
• Develop and conduct tests that evaluate and
monitor the equipment and personnel.
• Examine the test procedures themselves to ensure
their accuracy and reliability.

15. METHODS OF STERILIZATION OF STERILE
PRODUCTS
• Heat sterilization
▫ Moist heat (steam )
▫ Dry heat
• Gaseous sterilization
▫ Ethylene oxide
• Membrane filter sterilization
• Radiation sterilization
▫ Gamma
▫ Beta
▫ Ultraviolet
▫ microwave

16. QUALITY CONTROL TESTS FOR STERILE PRODUCTS
• The following tests are performed on the
finished sterile product to maintain quality
control :
▫ Leakage test
▫ Clarity test
▫ Pyrogen test
▫ Sterility test

17. Leakage test
• This test is performed by immersing the ampoules in a
vacuum chamber consisting of a dye like 1 % methylene
blue solution.
• A vacuum of about 27 inches Hg or more is created for
about 15 to 30 minutes.
• This negative pressure causes the solution to enter in the
ampoule with defective sealing.
• The vacuum is released and ampoules are washed
externally.
• The coloured solution in the ampoule confirms the
leakage and are discarded.
• High frequency spark test detects pin holes in the
ampoules is another means of detecting leakage.

18. Clarity test
• The parenterals must be free from microorganisms
or any particulate matter like glass,fibre,floaters.
• Particulate matter- defined as presence of
undesirable,extragenous,mobile and undissolved
substances within the sterile products.
• Methods to detect particulate matter:
▫ Visual method
▫ Microscopic count or membrane filtration method
▫ Light obstruction method
▫ Coulter counter method

19. Visual method
• The filled containers are examined by holding
the neck of the containers against illuminated
screen.
• Black surface is used for light coloured particle
and white is used for dark coloured particles.
• If any particle is visible, then that container is
discarded.

20. Microscopic count method
• A measured sample solution is filtered through a
membrane filter. The collected particles on the
surface of the filter are then counted with the
help of a microscope at 100X magnification.
• The whole method is carried out under aseptic
conditions

21. Light obstruction method
• The method uses an electronic instrument that
produces a light beam of high intensity.
• The solution is allowed to pass under this bright
light.
• If the particle present in the solution passes
through, a shadow cast is produced due to the
obstruction of light by the particles.
• The particles are measured and counted
automatically by the device.

22. Coulter counter method
• The electronic method is used to detect the
particles and also to determine the particle size.
• The sample solution is added to an electrolyte
solution.
• This solution is drawn through a small orifice of
the device.
• Positive and negative electrodes are present one
on either side of the device.

23. Continue..
• As the particles passes through the orifice, it displaces its
own volume of electrolyte and at same moment an
increase in electrical resistance is observed between two
electrodes.
• The resulting voltage pulses which are proportional to the
particle size are amplified and are measured and counted.
• Particles below 0.2 um can also be detected by this
method.

24. Pyrogen test
• Test involve measurement of rise in body temperature of
rabbits due to IV injection of sterile solution of substance
under examination Designed for products that can be
tolerated by test rabbits
• Test animal:
• Healthy, either sex, same variety, weight NLT 1.5 kg
• Diet- complete and balanced diet
• Condition- no loss of body weight during week preceding
the test
• Temperature- uniform
• Do not use the animal for pyrogen test repeatedly once
every 48 hr
• Temperature rise by 0.6 degree

25. Test:
• Use group of rabbits.
• Preparation of sample- Dissolve the substance in sterile
pyrogen free saline solution. Warm the liquid under
examination to approx. 38.5 degree before injection
• Procedure-
• a) Record temp of all animal at interval NMT 30min,
beginning at least 90 min before injection of solution
under examination and continue for 3hr after injection.
• b) Record the initial temperature of each rabbit (NMT
40min immediately preceding the injection). It should be
variats 0.2degree and not more than that, between two
successive animal.

26. • c) Do not use the animal having temperature more
than 39.8 and less than 38 degree.
• d)Inject the solution under examination slowly into
MARGINAL VEIN OF EAR of each rabbit over a
period not more than 4min. Amount of sample to be
use is given in monograph.
• e)volume of injection- NLT 0.5ml/kg and NMT
10ml/kg.
• f)Record the temperature of each animal at half
hourly interval for 3hrs after injection.
• g) The difference between initial temp and maximum
temp is taken to be response.
• h)when the difference is negative, the result is
counted as a zero response.

27. • Interpretation of result:
• Sum of response of group of 3 rabbits does not
exceed 1.4 degree and if the response of any
individual rabbit is less than 0.6 degree. The
preparation under examination passes the test.
• If the response of any rabbit is 0.6 or more or the
sum of response of three rabbits exceed 1.4, continue
the test using five other rabbits.
• If not, more than three of eight rabbit show individual
response of 0.6 or more and if sum of response of
group of eight rabbit does not exceed 3.7, the
preparation under test passes the test.

28. LAL (limulus amoebocyte lysate test)
• Test is used to detect endotoxins from gram negative bacteria.
• Also called as bacterial endotoxin test.
• principle:
• The test is based on the formation of a gel in the presence of
bacterial endotoxins and the lysate solution.
• The lysate consist of a proclotting enzyme and coagulogen
which are required for the reaction to occur.
• There are 3 main types:
• The gel clot test
• The tubidimetric test
• The kinetic chromogenic test

29. The gel clot test
• The lysate solution is mixed with an equal volume of the
test solution in a pyrogen free test tube.
• The test tube is allowed to stand for about 60 minutes.
now the tube is inverted and observed for the clot
formation.
• The presence of solid gel confirms the presence of
endotoxins.
• It requires positive (known concentration of endotoxin)
and negative (water) controls.
The tubidimetric test
• This test is based on the measurement of opacity change
in the LAL test due to the formation of gel clot.
• Opacity is directly proportional to the endotoxin
concentration.

30. The kinetic chromogenic test
• The test is based on the measurement of colour
change which is caused by the release of
chromogenic chemical, para nitroanilide.
• This p-nitroanilide is a by-product of the clotting
reaction during the LAL test.
• The quality of Para – nitroanilide produced is directly
proportional to the endotoxin concentration.
• ADVANTAGES OF LAL OVER RABBIT:
• Easy to perform
• Rapid
• Economical
• More sensitive

31. • Preparation of culture media:
• The culture media used for sterility test must be capable
of promoting the growth of a wide range of micro-
organisms such aerobic bacteria, anaerobic bacteria, and
fungi.
• Two types of media are used:
• Fluid thioglycolate medium and
• Soyabean casein digest medium
• Sampling:
• The selection of samples and the number of samples to be
taken from any given batch of sterile product.
• The material must be thoroughly mixed, if the sample is
to be taken from the bulk.
• The sample is taken at random from the batch of final
container.

32. • Determination of sample qty to be used in each
culture medium:
• IP has recommended the minimum qty of samples to
be used in each culture medium.
• Test methods:
Membrane filtration method
Direct inoculation method
• Membrane filtration method:
• Membrane filter of pore size not greater than 0.45 um
and diameter of about 47 mm that are effective in
retaining micro-organisms are employed for the test.
• The filtrations system and membrane are sterilized.
• The test substance are filtered through the membrane
under aseptic conditions.

33. • If the test substance has antimicrobial properties, the
membrane is washed with 3 successive 100ml
quantity of sterile diluent.
• The membrane is then aseptically cut into two equal
halves.
• One half of membrane is immersed in 100ml of soya
bean – casein digest medium and incubated at 20-25
C.
• The other half of the membrane is immersed in 100
ml of fluid thioglycollate medium and incubated at
30-35 C.
• Both the media are incubated for not less than 7 days.

34. • Direct inoculation method:
• The quantity of the test substance to be used in each
culture medium is directly inoculated into culture
media under aseptic conditions.
• Mix the inoculated liquid with the medium.
• If the test substance contains antimicrobial properties
then it is neutralized by adding suitable inactivating
substance to the medium.
• Incubate the inoculated media at 20-25 C in case of
soya bean-casein digest medium and at 30-35 C in
case of fluid thioglycolate medium for not less than
14 days.

35. • Interpretation of results:
• Examine the culture media during and after the
incubation period to detect the possible microbial
growth.
• The sample passes the test for sterility if microbial
growth is not found.
• If there is an evidence of microbial growth, the test
containers are kept aside. then repeat the test. if
growth is not found then the samples passes the test
for sterility.
• If microbial growth is found in the retest, isolate and
identify the organisms. If same organisms are found
as in the first test, the sample fails the test for sterility.

36. REFERENCES
• Pharmaceutical process validation,3rd edition, edited
by Robert A Nash, Alfred H Wachter, Marcel Dekker.