The document discusses the validation of various sterilization methods and water supply systems used in pharmaceutical manufacturing. It provides details on: 1. The key properties of sterile products and various sterilization methods like heat, gas, radiation. 2. The validation process for steam, dry heat and ethylene oxide sterilization including qualification of equipment and instruments, heat distribution studies, biological indicators, and establishing a monitoring program. 3. Types of water systems used, water treatment techniques, equipment components, design considerations for storage and distribution, and the concept of validation involving engineering design, operating procedures, maintenance and testing under all conditions.

1. Process validation of Sterilization
& Water Process Systems

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 variation in the rate of heating of the slowest heating
zone must be known, so this variation must be determined
under fully loaded conditions
• 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. • The final step in steam sterilization process the
establishment of a monitoring program to ensure
that the validated cycle remains essentially
unchanged in the future.
• Cycle monitoring usually involves the use of
thermocouples to measure heat penetration at the
cool spot location.
• Any changes in the load size, load configuration, or
container charecteristics (volume, geometry etc)
must be accompanied by repeat validation studies to
prove that the cool spot location has not changed
References: [10 ]

11. 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

12. • Heat distribution of 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 ]

13. 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.

14. 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 ]

15. 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 ]

16.  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 ]

17. 3.Biological process validation of dry heat
sterilization cycles
• mo’s known to be most resistant to dry heat must be used to
prove the ability of dry heat cycle to destroy them at the
coolest location in load. the dry heat process is claimed to
produce both sterile and pyrogen-free commodities, validation
studies must be done using both mo’s $ microbial endotoxins.
• Biological validation of dry heat cycles should be based on the
destruction of endotoxin rather than on the destruction of
mo’s because of the enormous dry heat resistance of
endotoxin compared to mo’s.
• With both mo’s $ endotoxin challenges, the cool spot
identified in heat distribution $ the heat penetration studies
will be the logical location to run the microbial challenge tests.
containers inoculated with the microbial cells or endotoxin will
be situated adjacent to identical containers into which
thermocouples are secured to monitor temp. References: [ 9 ]

18. 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]

19. 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.
temp is the easiest variable to measure $ monitor,
therefore temp is used as the indicator of the worst-case location
within the loaded Eto strilizer. Once the worst case location is
identified, the validation studies are conducted with the goal of
inactivating a known conc of indicator mo’s in the worst-case
location using a specific loading pattern with a specific Eto cycles.
References: [15]

20. 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.

21. 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]

22. 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]

23. VALIDATION OF WATER SUPPLY
SYSTEMS

24. OBJECTIVE
To understand:
1. The need for water quality manual
2. reason for usage of pharmaceutical water supply systems.
3. The technical requirements for water supply systems.
4. Different types of water supply systems.
5. Validation requirements.
6. Qualification & inspection requirement
References: [17]
24

25. INTRODUCTION
 High-quality water is essential for the manufacturing of
pharmaceuticals. Water is the most commonly used raw
material in pharmaceutical manufacturing.
 water is directly or indirectly used in the
pharmaceutical manufacturing such as a major
component in injectable products and in cleaning of
manufacturing equipment.
 It is one of the raw material that is usually processed by
the pharmaceutical manufacturer prior to use because it
cannot be supplied by the vendor.
Water is thus an important raw material in GMP and in
validating the manufacturing process.
References: [17] 25

26. INTRODUCTION
Quality of water should be specific for product quality.
Water contains,
• Organic and inorganic impurities
• Microbial contamination
• Endotoxin
• Particulate contamination
Low quality of water can lead to
 product degradation
 product contamination
 loss of product and profit
26
References: [17]

27. TYPES OF WATER
 Different grades of Water for Pharmaceutical Purposes-each
type has its on characteristic for all parameters.
Potable water
Purified water
Water for injection(WFI)
Sterile water for injection, inhalation, irrigation
Sterile bacteriostatic water for injection
References: [18]
27

28. References: [18]

29. DIFFERENT TECHNIQUES USED FOR
WATER TREATMENT
– De-chlorination (Sodium Bisulphite, Carbon Filter)
– Filtration
– Ultra Filtration
– Softening
– Demineralization
– Reverse Osmosis
– UV Treatment
– Deionization
– Ozonization
References: [18]
29

30. DIFFERENT EQUIPMENTS AND
COMPONENTS FOR WATER SYSTEM
• Piping
• Valves
• Pumps
• Pressure gauges
• Heat exchangers
• Distillation unit
• Filters
• Deionizers
• Sensors
• Auxiliary equipment
References: [19]

31. WATER STORAGE AND
DISTRIBUTION – CONSIDERATIONS
– Materials of Construction (Chemical and Heat
Compatibility)
• Stainless Steel (316 or 316L)
• Teflon, Silicone, Viton (gaskets, diaphragms)
– Minimize Dead Legs (<= 2 pipe diameters)
– Smooth Surfaces (Mechanical Polish , Electropolish)
– Clean joints (sanitary Tri®Clamp, automatic orbital
welding)
– Passivate interior surfaces to form barrier between water
and free iron (0.5 to 1% alkali at 160ºF for 30 minutes
followed by 1% Phosphoric Acid or Nitric Acid at 150ºF
to 180º F for 10 minutes.)
References: [19] 31

32. Conti….
• Design of the following should be appropriate to prevent
recontamination after treatment-
–Vent filter
–Sanitary overflow
–Tank UV light
–Conical Bottom
–Steam sterilization
• Combination of on-line (TOC, Conductivity meter etc.) and
off-line monitoring (lab testing by proper sampling) to ensure
compliance with water specification
32
References: [19]

33. VALIDATION CONCEPT
 To prove the performance of processes or systems under
all conditions expected to be encountered during future
operations.
 To prove the performance, one must demonstrate
(document) that the processes or systems consistently
produce the specified quantity and quality of water when
operated and maintained according to specific written
operating and maintenance procedures.
 validation involves proving-
1. Engineering design
2.Operating procedures and acceptable ranges for control
parameters
3. Maintenance procedures to accomplish it
References: [17]
33

34. Conti..
• the system must be carefully,
-designed
-installed
-tested during processing, after construction, and
under all operating conditions.
• Variations in daily, weekly and annual system usage
patterns must be validated.
References: [17]
34

35. STEPS OF VALIDATION
• Establishing standards for quality attributes
• Defining system and subsystem
• Designing equipment, control, & monitoring
technologies
• Establishing standards for operating parameters
• Developing an IQ stage & OQ stage
• Establishing alert and action levels
• Developing a prospective PQ stage
• Completing protocols and documenting each steps
References: [17]
35

36. Conti…
36

37. DESIGN QUALIFICATION OF WATER SYSTEM
Based on the URS, supplier designs the equipment.
• This is 1st step in the qualification of new water supply systems.
• Define process schematically by use of PFD and P&IDs.
• It is documented the design of the system & will include :
-Functional Specification.(Storage, purification, etc)
-Technical/Performance specification for
equipment.(requirements of water volume and flow, define
pumps and pipe sizes )
-Detailed layout of the system.
Design must be in compliance with GMPs and other regulatory
requirements.
References: [18]
37

38. REFERENCES
1. Mascoli , C,C should end product sterility testing continue,
med dev Diag ind 3: 8-9(1981)
2. Bowman. F.W.the sterility testing of pharmaceuticals. j.
pharm sci 58:1301-1308(1969)
3. Ernst R.R., West, K.L.Doyle, J.E.problemareas in sterility
testing .Bull parenter drug assoc 23:29-39(1969)
4. Akers ,M.J. In : parenteral quality control :sterility ,
pyrogen,particulate matter, and package integrity testing, 2nd
addition . Newyork: Marcel Dekker ,pp.1-4 (1994)
5. Brewer , J.H. In: G.L. Redish , ed. Anticeptics, disinifectants,
fungicides, and sterilization , 2nd ed. Philadelphia: Lea $
Febiger,pp,160-161(1957)
6. Food $ drug administration. Guidelines on general principles
of process validation. Rockville , MD:FDA(1984)

39. 7. Reich, R.R.,Whibourne, J.E.,Mcdaniel, A.W.effect of storage
conditions on the performance of B.steaothermophilus
biological indicators.
8.validation of steam sterilization cycles .technical monograph
no.1 parenteral drug association (1978) .
9.Tsuji,K.harrison,S.J.dry-heat destroction of lipopolysaccharide :
dry heat destruction kinetics.
10.pflug .I.J. holcomb .R.J principles of thermal destruction of
mo’s.
11.validation of dry heat sterilization used for sterilization and
depyrogenation, technical no.3parenteral drug
administration(1981)
12.simmons,P.L.validation of dry heat sterilizer.pharm eng
38(may- july 1981).

40. 13.simmons,P.L.Hot air and continuous sterilization.akers,M.J.,Ketron
,K.,Thompson. B.F.value requirements for the destruction of endotoxin
in the validation of dry heat sterilization cycles.
14.Avis,K.E.jewell,ludwig,J.D.avis,K.E.validation of a heating cellfor
precisely controlled studies on the thermal destruction of endotoxin in
glass.
15.Robertson,J.H.,Townsend.M.W,Allen;valenti,simmons,P.L.ETO
sterilization,caputo,R.A.Rohn.
16.ISO11137:1995.sterilization of health care products –requirements for
validation $ routine control –radiation sterilization .
17. ICH.GMP guide for active pharmaceutical ingredients.Q7A(march
15,2000).
18.general information:water for pharmaceutical
purposes.U.S.pharmacopeia.vol.25.rockville,MD:U.S.pharmacopeial
convention,pp.2261-2270(2002).
19.Pharmaceutical engineering guide vol.4:water $ steam guide. tampa,
FL:ISPE, (1997).

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