The document provides a comprehensive overview of sterilization, outlining its definition, validation processes, and the kinetics of microbial death. It details various sterilization methods, including thermal (e.g., steam under pressure, dry heat) and non-thermal (e.g., UV radiation, filtration), alongside principles for validating these processes. Additionally, it discusses related concepts such as sterility assurance levels (SAL), microbial inactivation rates, and the importance of cleaning and equipment validation in maintaining sterility in pharmaceutical applications.

2. Aim of Lecture :
Define Sterilization , explain the method of validation ,
mechanism and kinetics of microbial death’s through
following learning objectives.
Learning objectives:
• The students should define sterilization.
• Describe validation of methods.
• Describe microbial death kinetics.
• Describe methods of sterilization (thermal and non-
thermal).
• Describe mechanisms of sterilization methods.

3. Introduction
Sterilization is the process designed to produce a sterile state.
The traditional concept of the sterile state is the absolute
condition of total destruction or elimination of all living
microorganisms.
Principles of sterilization
• a sterile preparation is described as the absolute absence of
viable microbial contaminants.
• In practice, this definition is not achievable as a preparation
cannot be guaranteed to be sterile.
• Aseptic: is refer to the controlled process or condition in which
the level of MO contamination is reduced to the level that can be
excluded from the product during the manufacturing process.

4. Validation of Sterilization Processes
Different types of sterilization processes designed to
destroy or eliminate microbiologic contaminants
present in a product.
Validation of sterilization processes can be facilitated
by using quantitative, theoretically principles
such as: Microbial Death Kinetic Expressions.

5. Microbial Death Kinetic Terms
The number of organisms decreases exponentially with time
follow or approximate to first-order kinetics.
The death rate tells you what fraction of the initial population
survives a given period of exposure to the lethal agent.
Kinetics of cell inactivation

6. At which level Product is consider sterile??
Sterility assurance level (SAL), equal to or better than
10-6
Sterility terms:
1. Microbial inactivation rate constant (κ)
2. Decimal reduction value (D-value)
3. Inactivation factor (IF)
D value: microbial death kinetics for heat, chemical, and
radiation sterilization.
The D value is the time (for heat or chemical exposure) or
the dose (for radiation exposure) required for the microbial
population to decline by one decimal point (a 90%, or
one logarithmic unit, reduction).

7. - The decimal reduction time
- It is often more convenient to use the D-value instead of k
- The D-value is the exposure time required for the number of
survivors to change by a factor of 10
- It is a measure of the effectiveness of heat at any given
temperature

9. - D values defined for various M.O.
contained in certain environments
(liquids and solid surfaces) at specific
temperatures for heat sterilization and
at direct exposure to cobalt-60
irradiation.
- D values not be defined precisely for
M.O. exposed to such gases as ethylene
oxide because of the complex interaction
of heat, concentration of gas, and relative
humidity.
- But: D value estimated for gas
sterilization when it is possible to
keep heat and humidity values
constant, varying only the
concentration of gas

10. Inactivation Factor
Inactivation factor (IF) is the total microbial
inactivation by a lethal process
Defined as the reduction in the number of viable
organisms brought about by the lethal process:

11. Aseptic Processing
Aseptic processing also requires validation (to assure batch to
batch consistency in producing a given probability of product
sterility).
Probability of non-sterility levels can be obtained by
[process simulation testing] using: The percent
contamination level (% C) is calculated as follows:
1- Microbiologic growth medium
2- Suitable type and number of challenge microorganisms
3- Relevant number of containers.

12. Methods of sterilization
A- Thermal methods
1. Steam under pressure
2. Dry heat
B- Nonthermal methods
1. UV radiation
2. Ionizing radiation
3. Filtration
4. Gaseous

13. Thermal Methods
Lethal effectiveness of heat on M.O. depends upon:
• Degree of heat
• Exposure period
• Moisture present
Thermal methods of sterilization may conveniently
be divided into:
A- Dry heat
B- Moist heat

14. Notes
- Time required to produce a lethal effect is inversely
proportional to the temp. employed.
For example,
sterilization may be accomplished in 1 hour with dry heat at a
temperature of 170°C, but may require as much as 3 hours at a
temperature of 140°C
- The mechanism by which M.O. are killed by heat is the
coagulation of the protein of the living cell.
- The temperature required is inversely related to the
moisture present.

15. Dry heat sterilization
Substances that resist degradation at >140°C
may be rendered sterile by means of dry heat.
2 h at 180 °C or 45 min at 260 °C normally can
be expected to kill spores as well as vegetative
forms of all M.O.

16. In any heat sterilization process, the heating cycle includes
3 phases:
 Heating up stage : the thermal increment time for both the
chamber and the load of materials to be sterilised.
 Holding stage: the hold period at the maximum temperature
to achieve sterilization .
 Cooling stage: cooling period for the material to return to
room temperature.

17. The time to reach the stage of hold period is
affected by:
1. amount of material to be sterilized ( more time if
high amount)
2. The thermal conductance properties of martial
(the more poor conductance , the more time).
3. Heat capacity (the lower heat capacity, the longer
time).
4. Affected by moisture

18. Dry heat Sterilizer Types
A- Natural convection oven
Circulation depends upon the currents produced by
the rise of hot air and fall of cool air.
Disadvantages:
1- Easily blocked with containers, resulting in poor
heat distribution efficiency.
2- Differences in temp. of
20°C or more may be found
in different shelf areas.

19. B- Forced convection ovens
Provide a blower to circulate the heated air around the
objects in the chamber.
Advantages:
1- Efficiency is greatly improved over natural
convection.
2- Temp. differences at various locations on the shelves
may be reduced to 1°C.
3- The lag time of the load
material is reduced because
fresh hot air is circulated rapidly
around the objects.

20. C- Tunnel unit oven
With a moving belt, designed to thermally sterilize
glass bottles and similar items as they move through
the tunnel.

21. Dry heat sterilization is not suitable for Materials
such as:
1. Cellulose materials because they start to char at
more than 160°C
2. Many organic chemicals are decomposed by
elevated temp.
3. Rubber is rapidly oxidized
4. Thermoplastic materials they may melt

22. Due to the high temperatures required for dry
heat sterilization can only be used for:
Heat stable, moisture sensitive or moisture
impermeable pharmaceutical and medicinal.
These include products like;
1. Dry powdered drugs
2. Suspensions of drug in non aqueous solvents
3. Oils, fats waxes, soft hard paraffin silicone
4. Oily injections, implants, ophthalmic ointments,
ointment base

23. Advantages to provide dry glassware and metal
ware
1. Dry equipment and containers are essential in the
manufacture of an anhydrous product.
2. They are desirable to prevent dilution of an
aqueous product.
3. They can be kept sterile during storage more easily
than wet equipment.
4. Dry heat effectively destroys pyrogens, usually
requiring about twice the hold time for sterilization.

24. Moist heat sterilization
Steam under Pressure
o Moist heat is more effective than dry heat for thermal
sterilization.
o However, normal moist heat cycles do not destroy
pyrogens.
o Moist heat causes the coagulation of cell protein at a
much lower temperature than dry heat.
o The thermal capacity of steam is much greater than that
of hot air.
o At the point of condensation (dew point), steam liberates
thermal energy equal to its heat of vaporization.

25. o When saturated steam strikes a cool object and is
condensed, it liberates approximately 500 times the
amount of heat energy liberated by an equal weight of
hot air.
o Consequently, the object is heated much more rapidly
by steam.
o In addition, when steam under pressure is employed, a
rapidly changing fresh supply of heat- laden vapor is
applied to the object being heated.
o This is due both to the pressure under which steam is
applied and to the partial vacuum produced at the site
where steam is condensed, for it shrinks in volume by
about 99% as it condenses.

26. o This aids the penetration of steam into porous items such
as dressings.
o Wet saturated steam is less effective than dry saturated
steam as not as much condensation is produced and the
latent heat available is less.
o Superheated steam is another potential problem that must
be limited. Although it is hotter than dry saturated steam,
superheated steam is less efficient at releasing its heat to
cooler objects, as it is only as efficient as hot air at the same
temperature.
o Superheated steam condenses less readily than dry
saturated steam so less effective sterilant.

28. Moist heat is used for lower temperature sterilization
procedures. Temperatures of 100°C or lower [called
marginal or fractional methods].
A- Marginal (questionable reliability of the processes)
[This method of sterilization is reserved for
substances that must be processed by a thermal
method but cannot withstand higher
temperatures without degradation.
The assurance ofsterility is comparatively low].

29. B- Fractional
(processes are normally performed by two or three
exposures to moist heat, alternated with intervals
during which the material is held at room or incubator
temperatures).
Fractional methods of sterilization:
A- Tyndallization [temp. of 100°C]
B- Inspissation [temp. as low as 60°C]
(Are relatively effective in reducing the number of vegetative
forms of microorganisms, but are unreliable against spores).
Effectiveness improved by the inclusion of a
bacteriostatic agent.

30. Sterilization Indicators
Indicators are to validate sterilization process, also are place
where there is the greatest impediment to the penetration of
heat. e.g. for indicators :
- Thermocouple: These are connected to a recorder to
continuously record the temperature of the process
- Wax chemical pellets that melt at 121°C
- Paper strips that impregnated with chemicals that change
colour under the influence of moisture and heat.
- Biological indicators:
Resistant bacterial spores in sealed ampules or impregnated
in dry paper strips are used as biological indicators a
destruction of spores proves the sterilization cycle

31. Sterilization application with the use of moist heat under
pressure
1- Aqueous pharmaceutical preparations in hermetically sealed
containers [withstand temp. of autoclaving can be rendered sterile and
remain indefinitely unless tampering with the seal occurs].
Not applied: Non-aqueous preparations in sealed containers
(because no water is present within the container to generate
steam and thereby effect sterilization).
2- Equipment and supplies such as rubber closures, glassware, and
other equipment with rubber attachments; filters of various types; and
uniforms.
To be effective [air pockets must be eliminated that requires the items
to be wet when placed in the autoclave].
Note: when dry equipment is required and it must be sterilized
by autoclaving, the equipment may be dried in a vacuum oven
before use.

32. There are various types of autoclaves:
1- Simple non-jacketed
2- Downward displacement autoclaves
3- Porous load autoclaves

33. Non Thermal methods
Ultraviolet light
To get rid contamination in air and surfaces
within the processing environment.
It uses mercury vapour lamps to emit almost a
germicidal light at wave length 2537 angrsotm
(253.7nm)

34. Disadvantage of UV radiation:
sometimes the activity of microorganism recover after
exposure to Uv light.
The effectiveness of ultraviolet light depends on:
• Intensity of radiation and time of exposure
• Other factors: pH, temperature and humidity
• Type of contamination:
E.g., Intensity of radiation of 20 microwatts per cm2 requires:
 1,100 seconds exposure to kill B. subtilis spores
 and 275 seconds to kill S. hemolyticus

35. Ionizing radiation
High energy radiation emitted form:
• radioactive isotopes such as cobalt -60 (gamma rays)
• by mechanical acceleration of electrons at very high
velocities and energies ( cathode rays, beta rays)
Gamma rays are reliable.
Disadvantage:
Expensive and the emission can not be shut off unless it comes
from the mechanical source of accelerated electrons.
The accelerated electrons ( advantage ) providing higher and
more uniform dose rate output.

36. Filtration
• Removing of materials is usually by sieving.
• The filter is made of thin membrane composed of
plastic polymer (E.g., cellulose acetate and nitrate,
nylon and tyfoln)
• The most drawback is the solvent used, which might
destroy the filter membrane; however, as most
parenteral vehicle is water. So it is not an issue.
• Filtration should not remove the desired constituents

37. Mechanism of sterilization by filtration
1. Sieving: (main mechanism)
- Retaining material on the surface of the membrane.
2. Entrapment within the filter medium:
- ( very little) because the thicknesses is limited (it is only confined with
depth filter made of glass and paper).
3. electrostatic forces:
- Which (most common) in gas filtration due to probability of charge
generation due to flow of air and the dry conditions.

38. validation
Validation means rectification or confirmation.
Validation can be defined as a procedure that
demonstrates that a process under standard
conditions is capable of consistently producing a
product that meets the established product
specifications.

39. TYPES of validation
1) ANALYTICAL METHOD VALIDATION
2) EQUIPMENT VALIDATION
3) CLEANING VALIDATION
4) PROCESS VALIDATION

40. 1) ANALYTICAL METHOD VALIDATION
Method validation must prove that the analytical method used for a specific
test is suitable for which it is to be carried out.
TYPES OF PROCEDURES TO BE VALIDATED:
1) ACCURACY
2) PRECISION
3) REPEATABILITY
4) INTERMEDIATE PRECISION
5) REPRODUCIBILITY
6) SPECIFICITY
7) LINEARITY
8) DETECTION LIMIT
9) QUANTITATION LIMIT
10)ROBUSTNESS
11) RANGE

41. 2) EQUIPMENT VALIDATION
Equipment validation is to provide a high level of documented evidence that
the equipment and the process confirm to a standard.
TYPES:
(a)INSTALLATION QUALIFICATION
It ensures that all major processing and packing equipment and ancillary
systems are in conformity with installation specification, equipment
manuals ,schematics.
(b) OPERATIONAL QUALIFICATION
It is done to provide a high degree of assurance that the equipment functions
as intended.
It is conducted in 2 stages- component operational qualification & system
operational qualification.

42. (c) DESIGN QUALIFICATION
It is a documented review of the design, at an appropriate
stage in the project, for conformance to operational and
regulatory expectation.
(d) PERFORMANCE QUALIFICATION
It is a documented verification that all aspects of a facility,
utility or equipment perform as intended in meeting pre-
determined acceptance criteria.

43. 3) CLEANING VALIDATION
Cleaning validation ensures that there is no cross
contamination in a multi-product manufacturing plant and
also prevents microbial contamination.
TYPES OF CONTAMINATION TO BE CONSIDERED IN CLEANING
VALIDATION:
 Cross contamination
 Microbial contamination
 Contamination by cleaning or sanitizing agent
 Contamination by other agents

44. 4) PROCESS VALIDATION
Process validation is the means of ensuring & providing
documentary evidence that processes are capable of repeatedly
& reliably producing a finished product of the required quality.
TYPES:
(a) PROSPECTIVE VALIDATION
(b) CONCURRENT VALIDATION
(c) RETROSPECTIVE VALIDATION
(d) PROCESS RE-VALIDATION