VALIDATION OF STEAM

STERILISATION
 STERILISATION
Definition:
Sterilization can be defined as any process that effectively kills
or eliminates transmissible agents (such as fungi, bacteria, viruses)
from a surface, equipment, foods, medications, or biological culture
medium.
Different Techniques of Sterilization:
Physical Methods:
Dry heat Sterilization
Moist heat Sterilization
Radiation
Filtration.
Chemical Methods:
Gaseous Sterilization
Use of Disinfectants.
 VALIDATION
Definition:
Validation is the process of establishing documented evidence,
which provide a high degree of assurance that a specific process or
equipment will consistently produce a product or results meeting its
predetermined specifications and quality attributes.
If we want a pharmaceutical process to be validated then the
equipment used play major role in the whole process.
 PRINCIPLE
 Steam under pressure (saturated steam) is the most effective
means of sterilization for almost all items. To achieve sterility,
moist heat under pressure must come in contact with all surfaces
of all items for the appropriate length of time.
 Moist heat kills the organisms by coagulating and denaturing
enzymes and structural protein.
 Sterilization by moist heat of the most resistant spores generally
requires 121 0C for 15-30 minutes.
 Moist heat is used for the sterilization of culture media, and all
other materials through which steam can penetrate.
 VALIDATION OF STEAM
STERILISATION
The qualification–validation of a moist heat sterilization process
involves
● qualification of the autoclave by checking its performance against
the design specifications
● validation of the process by establishing the actual effectiveness
and reproducibility of the cycle in relation to the product and the
loading configurations
● assessment of possible changes in the product that could have
occurred during sterilization.
 PHYSICAL INDICATORS
• Qualification and Calibration of equipment
• Heat Distribution studies
• Heat Penetration Studies
• Pressure maintenance
To perform a proper validation, several items are required such as
•a temperature recorder (data logger) that can record and accumulate
temperature data collected by thermocouples
•a calibrated thermometer
•a computer that can analyze raw data and compute F value
•A laminar-flow hood
•Biological indicators (BIs)
•access to an incubator (55–60 C)
•a temperature bath and an ice bath for accuracy verification of the
thermocouples
Commissioning is the first step of qualification and consists of
obtaining evidence that equipment has been provided and installed in
accordance with its specifications and that it functions within
predetermined limits when operated as directed
by its instructions.
IQ–OQ phases:
•The IQ and OQ phases can be instituted separately or
simultaneously.
•During the IQ and OQ phases, various features of the autoclave are
examined and tested for proper functioning. This procedure includes
tests of
the chamber design
pressure vessel
door safety interlock system
Inspections of the chamber jacket, steam traps, electrical circuits
pressure and temperature indicators, vacuum pumps, vent filters, and
steam supply to the chamber.
•During the IQ phase, the calibration of all critical instruments—
process controllers, temperature sensors, pressure gauges, timers,
and measuring and recording instruments—should be performed.
•To monitor temperatures attained at various locations throughout the
chamber, thermocouples are used.
•The thermocouples are connected to computerized multichannel
recording systems that can record and print temperature data.
•For validation purposes, type T thermocouples are recommended
because they are stable throughout a wide temperature range.
•Depending on the chamber size, 10–20 thermocouples must be used
per cycle.
 Acceptance criteria:
The variation between the temperature of thermocouples and the
standard thermometer found to be within the acceptance criteria.
 Heat Distribution Studies
Carry out heat distribution studies by using a multi-point data
logger and maintain holding time for 15 minutes at 15 lbs.
Record the temperature and lag time of each probe
Acceptance criteria
All probes must reach temperature 121-124°C and pressure must
be within 15 to 18 lbs for 15min cycle.
 Load Pattern
Maximum Load
Load with all the glassware and media filled upto 70%, of the
chamber and the details are as follows.
250ml Conical flasks = 12 Nos with media, 13 Nos without
media, 500ml Conical flasks with media = 4nos, 1000ml Conical
flasks with media =4nos, Pipette10ml=10nos,Pipette 2ml=10nos,
Pipette 5ml=10nos, Pipette 1ml = 10nos,100ml
bottles=20nos ,Filtering unit=10nos, Test tubes =25nos
Minimum Load
Load with all the glassware and media required for a day’s
analysis (average).
250ml Conical flasks with media = 6nos, 500ml Conical flask
with media – 3 nos., 1000ml Conical flask - 1 Pipette 10ml=
10nos, Pipette 2ml= 10nos, Pipette 5ml= 10nos, Pipette
1ml=10nos, Bottles=10nos, Test tubes - 25nos.
Record the temperature and lag time
Performance qualification (PQ) phase:
PQ represents the confirmatory phase of the validation program and
consists of tests performed with the autoclave chamber under
loaded conditions.
During the PQ phase, which is sometimes referred to as
process validation, the following objectives must be attained:
● demonstration of the uniformity and effectiveness of the
process in inactivating or removing microorganisms to the
required safety level
● demonstration of the reproducibility of the process—through
the use of sufficient cycles
● demonstration of the compatibility of the process with the
items to be sterilized—through the assessment of the influence
of the sterilization process on the products.
Heat Penetration Studies
Carry out the heat penetration studies by using a multi point data
logger for the following loads mentioned. Record the temperature
and lag time if any as per annexure 1 and 2.
Acceptance criteria:
All 12 probes must reach temperature 121°C to 124°C and
pressure must be within 15 to 18 lbs. for 15 min cycle.
Microbial limit test :
Incubate the sterilised media flask or tubes from any one
Maximum and Minimum load of heat penetration studies and
observe for nutritive properties. Bacteria: 30 – 35 °C for 72 hrs,
Fungi : 20 – 25°C for 120 hrs
Acceptance criteria:
No microbial growth should be observed i.e. Negative control and
Nutritive properties of media must pass
Microbial challenge test :
Keep ampoules containing spores suspension of Bacillus
stearothermophilus 106 ­population at various location of the
autoclave along with probes and maintain the sterilisation
temperature at 15psi and 121°C during the heat penetration
studies, once on the maximum load.
 Acceptance criteria:
Autoclaved ampoules containing Bacillus stearothermophilus
spores suspension ampoules should not show any colour change
after five days of incubation.
 Heat penetration studies are considered the most critical
component of the entire validation program. These studies are
intended to find areas in the loads that are difficult to penetrate or
heat. When selecting the monitoring sites, one must take into
account the cold spots previously found during the monitoring of
the empty chamber. Heat penetration studies must demonstrate
the reproducibility of a cycle in relation to the loads and the
effectiveness of the killing effect throughout the chamber and
load.
CHEMICAL INDICATORS

It is based on the ability of heat to alter the chemical or physical

characteristics of variety of chemical substances. This change
should take place only when satisfactory condition for
sterilization prevails. Thus conforming that sterilization cycle
has been successfully completed chemical indicator generally
under go melting or color change.
Heat sensitive tape:
 Autoclave tape is an adhesive tape used in autoclaving to
indicate whether the correct temperature has been reached for
the elimination of virtually all living organisms (typically
121oCelsius).
 Small strips of the tape are applied to the items before
they are placed into the autoclave. The tape is similar to
masking tape but slightly more adhesive, to allow it to
adhere under the hot, moist conditions of the autoclave.
The tape typically has diagonal markings containing an
ink which changes colour (usually beige to black) upon
heating. One such ink contains 30.1% lead thiosulfate,
0.6% magnesium carbonate, 20.1% neocryl B8141,
30.1% ethanol, 22.7% ethyl acetate and 49% ink solids.

BIOLOGICAL INDICATORS
Bacillus stearo-thermophillus spores.
VALIDATION OF DRY HEAT
STERILIZATION
PARAMETERS VALIDATED BY USING
PHYSICAL INDICATORS
• Temperature Maintenance
• Heat Distribution of an empty chamber
• Heat Penetration Studies
• Air Balance Determination
 CHEMICAL INDICATORS
• Browne’s tube
The most commonly used chemical indicators for heat processes
are Browne’s tubes. These are small sealed tubes containing a
reaction mixture and an indicator. Exposure to high temperature
completes the reaction producing a change in the color of the
indicator. Exposure to high temperature completes the reaction
producing a change in the color of the indicator. All four
changes from red through yellow brown to green, the latter color
only being achieved after a specified time at the given
temperature.
 BIOLOGICAL INDICATORS
• Bacillus subtilus spores
 Spores of Bacillus subtilus in sealed ampoules of culture medium
are used for moist heat sterilization monitoring and these may be
incubated directly at 55 0C, thus may eliminate the need of aseptic
transfer (Table2).
 Aseptic transfer is also avoided by use of self contained units
where the spores strip and the nutrient medium are present in the
same device ready for mixing after use.
 The bacterial spores should have following qualities
 i. It should be non pathogenic
 ii. Should posses above average resistant to the particular
sterilization process.
 REFERENCES
 http://pharmtech.findpharma.com/pharmtech/data/
articlestandard//pharmtech/502002/40891/
article.pdf
 An Overview of the Validation Approach for Moist
Heat Sterilization, Part II B.M.Boca,
E.Pretorius,R.Gochin,R.Chapoullie, and Z.Apostolides
• I. R Berry, R. A Nash, Pharmaceutical process
validation, 2nd edi., Marcel Dekker Inc. New York,
revised and expanded, vol 57,351-368.
• Haider S I, Pharmaceutical Master Validation Plan,
The ultimate guide to FDA, GMP, GLP compliances,
St. Lucie Press, 2,17-21.