1.1 Verification and sequential operation of sterilization cycles
1.1.1 Test procedure: The prime objective of this test is to
verify the physical qualification and integrity of the Autoclave. A minimum of
three observations to be made.
1.1.2 Vacuum leak test
1.1.2.1 Objective : To check the sequential operation of vacuum leak test cycle
1.1.2.2 Procedure :
· Ensure that all the critical instruments are in
a calibrated state.
· Select the vacuum leak test cycle from HMI as
per SOP.
· During the vacuum leak test, the equipment should
operate as follows:
· Vacuum level reaches in the chamber and note the
reading (P1) by manual observation in PLC. (Negative pressure should
less than -0.85 bars).
· 2nd: Hold the vacuum for 5 minutes i.e. delay
before hold (Should not be more than 5 min and less than 2 minutes).
· 3rd: Note the reading (P2)
after 5 minutes delay period.
· 4th: Continue holding of vacuum
for further 10 minutes.
· 5th: Note the reading (P3) of
vacuum after the 10 minutes.
· Equipment the operator is to note the pressure
of P1, P2 and P3 from print out.
· Calculate the rate of vacuum drop as per the
following formula
Rate
of vacuum drop =
· Attach the cycle printout with
the identification of all sequential steps.
1.1.2.3 Acceptance criteria: The maximum acceptable leak should be less
than 10mm of Hg/ 10 minutes of hold.
1.1.3 Bowie-dick test.
1.1.3.1.1 Tools required: Bowie-Dick test pack.
1.1.3.1.2 Objective: The Bowie-Dick Test (Air removal test) is to evaluate the
efficiency of air removal during the pre-vacuum phase of the sterilization
cycle. The Bowie-Dick test is a diagnostic test of Autoclave's ability to
remove air from the chamber and prevents residual air from remaining in
porous masses.
1.1.3.2 Procedure :
·
Collect one Bowie-Dick
Indicator and record the details of autoclave and date.
· Place the Bowie-Dick indicator withstand near
the drain in autoclave empty chamber trolley.
· Select the 'Bowie-Dick Test' program from the
autoclave HMI and start the cycle.
· Collect the exposed Bowie-Dick Indicator
and autoclave print out after completion of cycle and attach to protocol.
1.1.3.3 Acceptance criteria: The indicator shall show a uniform change
after exposure to saturated steam at 121.1ºC to 123.9ºC
for 10 minutes.
1.2 Empty chamber distribution studies:
1.2.1 Tools required: T- type thermocouples or RTD
sensors, Datalogger and Dry bath.
1.2.2 Test procedure: The objective of the empty chamber heat
distribution study is to demonstrate the uniformity and stability of the
Autoclave medium through the Autoclave by mapping the temperatures at different
locations and to locate the cold and hot locations within the chamber.
1.2.2.1 Temperatures at various locations inside
the autoclave without load will be logged using pre-calibrated T- type
thermocouples by the Data logger system.
1.2.2.2 The empty chamber heat distribution study
shall be carried out in triplicate runs to demonstrate the consistency of the
system.
1.2.2.3 Create a standard set up for autoclave as per
the respective SOP.
1.2.2.4 Perform the pre-calibration of thermocouples or
RTD sensors as per the SOP.
1.2.2.5 Pass the probes to the autoclave chamber through
the validation port carefully and seal the port properly to avoid any leakage.
1.2.2.6 Fix minimum of 18 pre-calibrated thermocouples
(Type T) or RTD sensors inside the chamber by considering the locations like
chamber drain, temperature sensors other than drain, in the corners and the
geometric center of Autoclave, etc.
1.2.2.7 Use Teflon tape to secure thermocouples or
RTD sensors in position. The thermocouple or RTD sensors tips shall be
suspended to avoid contact with the internal surface of the Autoclave.
1.2.2.8 Connect the data logger and scan the time
and temperature thermocouples during study at regular intervals of 15 seconds.
1.2.2.9 Monitor the heat controlling temperature
sensors of Autoclave during the validation cycle for information.
1.2.2.10 Set the autoclave to a predetermined temperature
and other parameters by following the standard operating instructions.
1.2.2.11 Perform the requalification run by using the
created setup for autoclave and data logger as described in SOP.
1.2.2.12 Once the sterilization cycle completed, collect
the autoclave print out and generate the requalification study report from data
logger.
1.2.2.13 Check the maximum temperature, minimum
temperature, and temperature difference of max and min. temp for all Heat
distribution study probes generated from the data logger. Check the accumulated
lethal values for all thermocouples.
1.2.2.14 Chamber Come-up time: Time is taken by the
chamber i.e. heat distribution thermocouples to reach the minimum sterilization
the temperature of 121ºC.
1.2.2.15 The cold location identified during performance
qualification study, that same location shall be identified during this
requalification.
1.2.2.16 After completion of cycle collect condensate
sample from chamber to check bioburden and BET as per approved test procedures.
1.2.3 Acceptance criteria: The heat distribution study shall provide
typical acceptance criteria as per the following:
1.2.3.1 The temperature at each temperature-mapping the
probe should be within the range of 121.0ºC to 124ºC and pressure should be
1.2±0.1 kg/ cm2 during the sterilization hold
period. Throughout the dwell time, the temperature measured at a single
probe does not fluctuate by more than 1ºC.
1.2.3.2 Throughout the dwell time, each inbuilt
autoclave temperature sensor shall not differ more than ±2ºC for adjacent
temperature probe.
1.2.3.3 Coldest locations shall be consistent and
identified.
1.2.3.4 Condensate shall meet WFI specification for
microbial & BET limits.
1.2.3.5 Overall performance characteristics like a
vacuum and pressure levels, overall cycle time shall meet cycle specifications
and are essentially equivalent.
1.3 Validation plan for loaded: The following validation studies shall be
carried out to
establish
the sterilization cycles for Autoclave.
1.3.1 During performance qualification, perform 3 runs
to each minimum & maximum load.
1.3.2 In case of requalification three runs shall be
performed for each minimum & maximum load.
1.4 Loaded chamber heat distribution studies
1.4.1 Tools required: T- type thermocouples or RTD
sensors, Datalogger, and Dry bath.
1.4.2 Objective:
The objective of this study is to establish that appropriate
temperatures exist within (and adjacent to) the load to effect adequate
sterilization and also to locate a cold spot in the chamber loaded with
specified load configuration in a specific pattern.
1.4.3 Procedure
· The test shall be carried out 3 runs to obtain
consistent results during performance qualification or requalification.
· Load the autoclave with specified loading
configuration and pattern.
· Fix pre-calibrated probes at the same locations,
as we're in the empty chamber
· Place one T- type thermocouple or RTD
the sensor in the drain, adjacent to the sensor that controls the chamber
temperature
· Use Teflon rope to secure sensors in position.
Ensure that the tips of T- type thermocouples or RTD
sensors do not touch any metallic surfaces of the autoclave.
· Set sterilization cycle parameters and operate
the autoclave as per SOP.
· Record the temperature of the entire T-
type thermocouples or RTD sensor throughout the sterilization cycle at
intervals of 15 seconds.
· Locate the coolest point for each type of load
in a specific loading configuration and pattern.
1.4.3.1 Acceptance criteria:
1.4.3.2 The
loaded chamber heat distribution temperatures uniformity during the
sterilization period (dwell time) shall be within 2ºC of the mean chamber
temperature.
1.4.3.3 The difference between the maximum and minimum
temperature (ΔT) within the heat distribution thermocouples (except drain
point) at any time point shall not be more than 1ºC.
1.4.3.4 The temperature difference between the autoclave
inbuilt probes shall be within 2ºC to the adjacent temperature probe.
1.5 Heat penetration studies with biological
indicators:
1.5.1 Tools required: Calibrated T type thermocouples or RTD probes,
Dry bath, silicon sealant and Datalogger.
1.5.2 Objective:
1.5.2.1 Heat distribution: To perform the temperature mapping throughout
the sterilization autoclave with load conditions and to locate the cold and hot
locations in the chamber for the respective load configuration.
1.5.2.2 Heat penetration: The steam is sufficiently penetrating the
inner parts of items for sterilization and to achieve a minimum 12 log
reduction (6 log reductions for media loads) after completion of the
sterilization cycle at the specific set temperature in dwell time.
1.5.2.3 Biological indicators challenge study: The steam sterilization process, when
challenged with Geobacillus Stearothermophillus biological indicator spore
strips (ATCC 7953(1)) having minimum spore population of 106 spores/strip,
should reduce the bacterial load by 12 log reduction for heat resistant
loads, whereas 6 log reduction for heat-sensitive loads.
1.5.3\ Procedure:
1.5.3.1 The loaded autoclave HD & HP with biological
indicators challenge study shall be carried out for all types of load
configurations and load patterns individually.
1.5.3.2 The combination studies can be carried out with
single run for every six months and triplicate runs during requalification.
1.5.3.3 Ensure that empty chamber HD study is completed
before carrying the loaded chamber HD & HP studies.
1.5.3.4 Fix pre-calibrated thermocouples (Type T) or
RTD’s within the chamber in different locations for mapping the distribution
temperatures with the load by considering locations which are monitored in
the heat distribution study during performance qualification
Study. 1.5.3.5 Place the items as per the load patterns cycles and shall be
performed with minimum and maximum loads.
1.5.3.6 Pass the probes into the chamber through
validation port and seal the port properly to avoid any leakage.
1.5.3.7 The heat distribution probe locations placed
accordance to 'Probe Placement record’ and shall be recorded.
1.5.3.8
Use Teflon tape to
secure thermocouples in position. The thermocouple tips shall be suspended to
avoid contacting any solid surfaces.
1.5.3.9 Place the autoclave in-built probes in different
locations as shown in the in-built probe placement diagram and shall be
recorded in probe placement record.
1.5.3.10 One Probe shall fix in the drain line to monitor
the steam condensate temperature.
1.5.3.11 Heat penetration study: Load the items into the
autoclave as described in the section.
1.5.3.12 Place minimum 10 pre-calibrated heat penetration
thermocouples (Type T) or RTD’s within the load as described in the 'Probe
Placement Drawing" to monitor the penetration temperatures and container
mapping.
1.5.3.13 Record the heat penetration probe location
details in 'Probe Placement Record'.1.5.3.14 Biological indicators challenge study:
1.5.3.15 Place the biological indicator strips of
Geobacillus Stearothermophillus (ATCC 7953(1)) within the load just
adjacent to the heat penetration mapping thermocouple.
1.5.3.16 Place one Biological indicator strip in the
drain point just adjacent to the drain point monitoring thermocouple.
1.5.3.17 Set the Autoclave parameters as per standard
operating procedure.
1.5.3.18 Operate the Datalogger as per the respective
operation SOP.
1.5.3.19 Operate the sterilization autoclave as per the
SOP.
1.5.3.20 Start recording the thermocouple readings like
temperatures and accumulated lethality at every 15 seconds during the complete
sterilization cycle.
1.5.3.21 Stop the cycle once the sterilization cycle
completed and collect the autoclave printout.
1.5.3.22 After completion of sterilization cycle collect
condensate sample from chamber to check bioburden and BET as per approved test
procedures.
1.5.3.23 Collect the biological indicators after
completion of a cycle and inoculate into the medium and perform the study as
described in the General Test Procedure.
1.5.3.24 Calculate the maximum, minimum, average
temperatures within the probes during the sterilization phase for Heat
distribution study.
1.5.3.25 Check the accumulated lethal values for all Heat
Penetration thermocouples of the specific loading configuration generated
from the data logger and establish the set temperature and sterilization time
based on the study.
1.5.3.26 Chamber come-up time: Time taken by the chamber
i.e. heat distribution thermocouples to reach the minimum sterilization temperature
of 121.1ºC.
1.5.3.27 Load Come-up time: Time is taken by the load
i.e. heat penetration thermocouples to reach the minimum sterilization
temperature of 121.1ºC.
1.5.3.28 Heating lag: Heating lag is the difference
between the load come-up time and chamber come-up time.
1.5.3.29 Calculate the Spore Log Reduction (SLR) &
Sterility Assurance Level for all sterilization hold time of the cycle.
1.5.4 Acceptance criteria:
1.5.4.1 The temperature at each temperature-mapping the
probe should be within the range of 121.0ºC to 124ºC and pressure should be
1.2±0.1 kg/ cm2 during the sterilization hold
period. Throughout the dwell time, the temperature measured at a single
probe does not fluctuate by more than 1ºC.
1.5.4.2 All heat penetration probes shall accrue 12 log
reduction for heat resistant loads whereas 6 log reduction for heat sensitive
loads.
1.5.4.3 The sterility assurance level (SAL) obtained
shall be a min of 10-6 for heat resistant loads.
1.5.4.4 The come-up time of load, chamber and heating
lag are essentially equivalent in all the runs with variation of NMT ±1 minute.
1.5.4.5 The sterilization cycle parameters like dwell
time, pressure and vacuum levels shall meet the cycle specifications and
essentially equivalent for all 3 runs during requalification.
1.5.4.6 All biological indicators exposed to the
sterilization process shall be negative for growth after incubation.
1.5.4.7 Condensate shall meet WFI specification for
microbial & BET limits.
1.6 Estimation of F0 Value:
1.6.1 Objective: To identify no. of equivalent minutes of
sterilization at temperature 121.1°C delivered to a container or unit of
product calculated using a Z-value of 10°C temperature.
1.6.2 Test Procedure:
1.6.2.1 Attach all the data prints of the cycles from
autoclave and data logger to the respective data sheet.
1.6.2.2 Calculate the F0 value at each
temperature mapping probe for each time interval during dwell period by using
the following f
F0:
∑ 10 (T-121.1)/z Δ t
F0 value is time that is
equivalent to the sterilization effect at 121.1ºC.
T = Product temperature at specific time
in ºC
Δ
t = Time interval between temperature measuremen
Z
= Temperature required to change the D value by a factor of 10ºC
D
value is time required to reduce a microbial population by a
one- logarithm
reduction
Note: If
the Δt is 15 seconds and Z value is 10ºC for sterilization study so the final
formula for calculation of Fo values is:
Fo = ∑10 (T-121.1)/10×0.25
1.6.2.3 Calculate cumulative Fo value by
summing the Fo values for every 15 seconds obtained from 90ºC
during heat-up phase, sterilization hold period and up to 90ºC of cooling phase
by using the following formula:
F0 =
∑ (sum of lethal rates)
1.6.3 Acceptance Criteria: The calculated minimum F0 value
shall be more than biological Fo value (Biological F0 = D(Log
A – Log B)
Where D =
Thermal decimal Time
Log A = Population of biological Challenged indicator
Log B = Sterility Assurance level
1.7 Estimation of Sterility Assurance Level (SAL)
and Spore log reduction (SLR):
1.7.1 Objective: To demonstrate the degree of process lethality in terms of
Sterility Assurance Level (SAL) for the sterilization of autoclave for
individual load configuration.
1.7.2 Test Procedure:
1.7.2.1 Sterilization Assurance Level is the expected
probability of an item being non-sterile after exposure to a valid
sterilization process.
1.7.2.2 SAL is a level of microbial inactivation is
described by an exponential function.
1.7.2.3 PNSU indicates that the probability of a single
viable microorganism being present on sterilized item is one in one million
after the item has undergone 6 log reductions.
1.7.2.4 A microbial survival probability (SAL) of 10-6 is
considered for steam sterilization process for heat resistant loads.
1.7.2.5 The SAL calculation can be done by using
following equation: Identify the minimum F0 value obtained from
the study and calculate the SLR and SAL values as follows:
1. SLR = Sum of F0 / D value
2. SAL = 10 log of BI population - SLR
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