This document discusses the validation of various sterilization processes including steam, dry heat, ethylene oxide, and radiation sterilization. It provides details on qualification and calibration of equipment used, selection and calibration of biological indicators, and heat distribution and penetration studies. The key steps in validating dry heat, ethylene oxide, and radiation sterilization cycles are also summarized.

1. Process validation of Sterilization &
Water Process Systems
GUIDED BY Presented by
Dr. K. Kishore Kumar Ms. V. Gouthami
256213886029

2. STERILIZATION VALIDATION
• Sterile products have several unique properties such as
1. Free from micro organisms
2. Free from pyrogens
3. Free from particulates
4. High standards of purity and quality
References: [1,2,3, & 4]

3. Methods of sterilization of products
HEAT:-
1. Moist heat-auto clave
2. Dry heat-hot air oven
GAS:-
1. ethylene oxide
2. Peracetic acid
3. Hydrogen peroxide(vapor phase)
4. Chlorine di oxide
References: [5& 6]

4. Radiation
1. Gamma rays
2. Beta rays
3. Ultraviolet
4. Microwave
References: [5& 6]

5. Validation of steam sterilization cycles
Qualification and calibration
1. Mechanically checking ,upgrading, and qualifying the
sterilizer unit

6. Selection and calibration of thermocouples
• Cu constantan wires coated with teflon are a popular choice as
thermocouple monitors
• Accuracy of thermocouples should be ±0.5°C. Temperature accuracy is
especially important in steam sterilization validation.
• Thermocouple accuracy is determined using NATIONAL BUREAU OF
STANDARDS (NBS) traceable constant temperature calibration
instruments.
• Thermocouples should be calibrated before and after validation
experiment at 2 temperatures i.e. 0C & 125 C .
• New thermocouple-recording devices are capable of automatically
correcting temperature
• Any thermocouple that senses a temperature of more than 0.5 C away
from the calibration temperature bath should be discarded
• Temperature recorders should be capable of printing temperature data in
0.1°C increments
References: [8 & 9]

7. Selection & calibration of BI
• The organism most resistant to steam heat is the
bacterial spore B. stearothermophilus. This bacterial
spore is commonly used BI’s in validating steam
sterilization cycles.
• Spore trips or spore suspensions are used in the
validation studies. the no. of mo’s per ml of suspension
must be as accurately known as D value.
• Precautions should be taken to use proper storage
conditions for B. stearothermophilus BIs .storing in the
freezer provides a more stable resistance profile for the
shelf life of the indicator.
References: [7 ]

8. Heat distribution studies
It include 2 phases
1. Heat distribution in any empty autoclave chamber.
2. Heat distribution in a loaded autoclave chamber.
a. 10-20 thermocouples should be used/cycle. thermocouples
should be secured inside the chamber.
b. The trips where the wires are soldered should not make contact
with the autoclave interior walls or any metal surface.
c. 1 end of thermocouple should remain in an ice bath and high
temperature oil bath during each cycle for reference when the
temp monitoring equipement has the capability for electronically
compensating each temp measurement against an internal
reference.
d. Heat distribution studies following the initial study may employ
fewer thermocouples as the cool spot in the chamber & in the
load is identified.
e. The difference in temp b/n the coolest spot $ the mean chamber
temperature should not be greater than  2.5C.
References: [8 ]

9. Heat penetration studies
• This is the most critical component of the entire
validation process
• Thermocouples will be placed both inside and outside
the container at the coolspot location(s) in the steam
exhaust line and in constant temperature baths outside
the chamber
• The sterilization cycle design must be based on the
heating charecteristics of the load and containers
located in the slowest heating zone of the load.
• The effect of load to load variation on the time-temperature
profile must also be determined.
• Then the statistically worst case conditions should be
used in the final sterilization process design

10. Validation of dry-heat sterilization
cycles
1. Batch oven validation
• Air balance determination in an empty oven
data are obtained on the flow rates of both
intake and exhaust air. air should be balanced
so that positive pressure is exerted to the
non sterile side when the door is opend and
air velocity across and up and down the
opening of the door is ±50 FPM of the
average velocity

11. • Heat distribution in an empty chamber
thermocouples should be situated according to a
specific predetermined pattern. Repeatability of
temp attainment and identification of cold spot
can be achieved if the temp range is ±15°C at all
monitored locations.
• Heat penetration studies. These studies should be
designed to determine the location of slowest
heating point within a commodity at various
locations of test load in sterilizer.
• Mechanical repeatability. during all these studies
mechanical repeatability in terms of air velocity,
temp consistency, reliability and sensitivity of all
the oven and instrumental controls must be
verified.
References: [11 ]

12. 2.Tunnel sterilizer validation
 Air balance determination
• Proper air balance is more critical to a tunnel sterile
process than a batch oven process .since the items being
sterilized are exposed to a different air systems(eg:-heating
zone $ cooling zone).in the absence of a critical balance of
air dynamics, either the items will not be cooled sufficiently
once they exit the tunnel or they will be cooled too quickly.
causing the glass to shatter and contaminate the entire
tunnel area with particles.
• The major problem in validating tunnel sterilizers is the
control of particules. not only are items exposed to great
extreams in temp, but also the conveyer belt is a natural
source of particulates because metal is moving against
metal.
• Air must be particulate-free as it enters the tunnel area;
therefore, all high efficiency particulate air(HEPA)filters in
the tunnel must be tested and certified prior to validation
studies.

13. Heat distribution studies
• Thermocouples used in tunnel sterilizer validation
must be sufficiently durable to withstand the
extremely high( ≥ 300 c)temperatures in the
heating zone area of the tunnel heat-distribution
studies should determine where the cold spots
are located as a function of the width of the belt
$ height of the tunnel chamber. trays or tracks of
ampules are vials should run through the tunnel
• Bottle-mapping studies may also be conducted
during this phase. the purpose of these studies is
to determine possible locations inside the
container that are most difficult to heat.
References: [12 ]

14. Heat penetration studies
• For testing of the tunnel sterilization, heat-penetration
studies must be completed in order to identify the coolest
container in the entire load. Results of heat-distribution
studies should aid in the predicting where the coolest
location with in the load should be. Thermocouples should
be diposited at or near the coolest point inside the
container from bottle-mapping studies.
• The containers inner surface should be in contact with the
thermocouple tip because the objective is to sterilize the
inner walls of the container, as well as the inner space.
• Every loading should be done using 10-20 thermocouples
distributed through out the load.
References: [ 9 ]

15.  Mechanical repeatability
• Air velocity, air particulates, temp consistency
and reliability of all the tunnel controls(heat
zone temperatures, belt speed, and blower
functions)must be proved during the physical
validation studies.
References: [ 9 ]

16. Step by step sequence in the microbial validation of a dry
heat process for sterilizing and depyrogenating large volume
glass containers by wegel $ akers et al
1. Place spore carrier in approximately 12 glass bottles located at the
coolest area of the oven. bottles adjucent to the inoculated bottles
should contain thermocouples for the monitoring purposes .
2. Run a complete cycle using the desired loading pattern for future
dry heat overkill cycles.
3. After the cycle, aseptically transfer the spore strip to vessels of
culture meedia. if spore suspensions were used, aseptically transfer
the inoculated bottles to a laminar air flow work station $ add
culture media to the bottles. use approximate possitive $ negative
controls
4. Determine the no. of survivors by plate counting or fraction
negative methods.
References: [11]

17. Validation of ethylene oxide sterilization cycles
Eto has been a sterilant for over 50 years.
• 5 variables critical to the Eto process. they are
1. Eto concentration
2. Relative humidity
3. Temperature
4. Time
5. Pressure/vaccume.
References: [15]

18. Procedure for the Eto cycle validation
1. Use a laboratory sized Eto sterilizer during early phases of the
validation process as long as the sterilizer is equipped with
devices allowing variability in vaccume ,relative humidity, temp,
gas pressure, timing,$ rate of gassing the chamber.
2. Verify the calibration of all instrumentation involved in
monitoring the Eto cycle.
3. Perform an extensive temp distribution study using an empty
sterilizer.
4. Do a series of repetitive runs for each sterilization cycle in an
empty vessel in order to verify the accuracy and reliability of the
sterilizer contorls and monitoring equipment.
5. Do a series of repetitive heat distribution and heat penetration
runs using a loaded Eto sterilizer.

19. 7.Test should be conducted on the final packaged product.
8.Institute a documented monitoring system primary relying on
bio-logical indicators,with lesser reliance on end-product
sterility testing.
References: [15]

20. Validation of radiation sterilization process
• The major objective in validating a radiation sterilization
process regardless of whether the mode of radiation is
cobalt-60,cesium-137 or electron beam.
• The radiation sterilization cycles are validated based upon
the achievement of sterility ,many factors must be
considered in the utilization and approval of the radiation
sterilization process. such factors include
 The physical appearance of the container system and its
contents,
 Stability of the active ingradient, if present, and
 Safety of the irradiated material.
References: [16]