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INDUSTRIAL OPERATION AND VALIDATION OF A DOUBLE DOOR

AUTOCLAVE

PRESENTED BYB.KARTHIKM. Sc MICROBIOLOGY

INTRODUCTION OF AN AUTOCLAVE:-

An autoclave is a pressure chamber used to carryout industrial

processes requiring elevated temperature and pressure different

from ambient air pressure.

It is used for the sterilization of media, glass were, gloves,

gowns etc.,

It is a moist type of sterilization unit.

Normal temperature and pressure required for autoclave is

121 C and 15lbs pressure. ⁰

Aim:- To operate and validate the industrial double door autoclave.

(Pharmalab)

Objective:- Operate and Validate the industrial double door auto clave.

Dehydrated media, glass were and accessories sterilization for

analysis .

HOW DOES AN AUTOCLAVE WORKS

• Steam enters the chamber jacket, passes through an operating valve and enters the rear of the chamber behind a baffle plate. It flows forward and down through the chamber and the load, exiting at the front bottom. A pressure regulator maintains jacket and chamber pressure at a minimum of 15 lbs, the pressure required for steam to reach 121ºC (250ºF). Overpressure protection is provided by a safety valve.

WHAT IS VALIDATION ?

• Validation shows that a process (or item of equipment) does what it is claimed to do.

• Why validate? – Need to show that sterile equipment is being taken

into sterile core - do not want to contaminate production (which could lead to adulterated product)

– For micro testing (both bacteriological and Viable Environmental Monitoring), using sterile equipment helps reduce false positives.

– Need to show that decontamination cycles are effective

HOW TO VALIDATE

• The usual – URS. IQ, OQ, PQ.

– This discussion will focus on PQ.

• IQ: does equipment meet the URS requirements? Is everything that was

on the box, in the box? Is the unit installed properly. Are support

programs in place for ongoing operation of A/C?

• OQ: does the A/C operate properly? Does the unit hold temp and pressure

correctly?

• PQ: validation of autoclave cycles and loading patterns – need to show

sterilization.

• What is D value?– Refers to decimal reduction time - The time required at a

certain temperature to kill 90% of the organisms being studied. Thus after an organism is reduced by 1 D, only 10% of the original organisms remain. Dependant on microbe and initial numbers. E.g. D value of 1.5 = 1CFU .

• What is Z value?– Refers to the temperature change required to produce a 1

log reduction in D value.• What is F0?– The number of minutes to kill a specified number of

microbes with a Z value of 10oC at a temp of 121.5oC.– Often confused with the time the chamber is held at

elevated temperature and pressure and in practice is the same thing.

• Overkill– Use many more microbes than would find

on items typically autoclaved. Negates the need to test sample for bio load before running the cycle.

– Use a sterilisation time exceeding what is necessary to kill a large number of microbes. Negates the need to determine D value of microbe.

Procedure to operate an autoclave :-

Switch on auto clave

Fill the water tank with water line

Switch On the heater switch

Open the door – 1

Load the chamber with media or glass were or with accessories

Arrange the sensors as we required

Close the door- 2

login with user id and password

Select the type of recipe

Start the cycle

Set up the strip chart

Observe the temperatures on the display

After completion of cycle open the door – 2

Give print to the printer

Calculate the F0- value, D-value and Z- value

with the use of computer software

Recipes for validation of a double door autoclaveThere are mainly 5 recipes present in the validation of a double

door auto clave.

They are :-

1. VLT (Vacuum leak test)

2. Bowie – dick test cycle

3. Steam In Process (SIP)

4. Gravity test cycle

5. HPHV test cycle

Vacuum leak test:-

Vacuum leak test is performed to check any leakage in

autoclaves. This test is also called chamber integrity test. In

VLT, first vacuum is applied inside the chamber and then wait

for the vacuum to reach at set vacuum point then hold the

vacuum for required time and when hold time completed

vacuum breakage occurs. After that check the results whether

autoclave leakage is within the limits or not.

PARAMETERS:-

Chamber level test vacuum :- -0.007bar

Stabilization time :- 5min

Vacuum hold time :- 10min

Acceptable leakage:- 0.0013bar/min

Print interval :- 60sec

Frequently test :- 15 days ± 2 days

Acceptable criteria :- 0.0013 bar/Min

No. of cycles run for validation :- 3

Bowie Dick test :- This is the cycle done for the removal of contaminated air

pockets in an autoclave.

In this the chemical indicator Bowie Dick

chemical indicator papers will be used.

The Bowie Dick indicator paper will turn from yellow to

black(or) brown in colour.

Bowie – Dick test papers

Parameters :-Pre vacuum :- -0.700Bar Pre vacuum hold time :- 01 minPre pressure :- 0.300BarPre pressure hold time :- 01 minHeat up 1 :-107 C⁰Heat up 1 hold :- 01minHeat up 2 :- 112⁰Heat up 2 hold :- 01minHeat up 3 :- 115⁰Heat up 3 hold :- 01 minHeat up 4:-117⁰Heat up 4 hold :- 1minSterile hold temp :- 121.3⁰

Control band :- 000.3Sterile hold time :- 11 minSterile hold reset :- 120.6⁰ Sterile hold stop count :- 121⁰Post vacuum :- -0.700BarPost vacuum hold time :- 02 minDoor open temp :- 90Print interval :- 60 secControl temp :- min for all

SIP (Steam In Process) cycle:-

It is the cycle done for vent filter sterilization of an autoclave.

It is the test done once per a month

Acceptance criteria :-

For SIP of filter temperature should be more than

121 C during sterilization.⁰

Parameters :-Heat up 1 :-107 C⁰

Heat up 1 hold :- 01min

Heat up 2 :- 112 C⁰


Heat up 3 :- 115 C⁰

Heat up 3 hold :- 01 min

Heat up 4:-117 C⁰


Sterile hold temp :- 122 C⁰

Sterile hold time :- 15 min

Sterile hold reset :- 120.6 C⁰

Sterile hold stop count :- 121 C⁰

Sterile temp over shoot :- 130 C⁰

Post vacuum :- -0.700Bar

Post vacuum hold time :- 02 min

Door open temp :- 90

Print interval :- 60 sec

Gravity cycle :- This is the cycle used for the sterilization of the media and water.

This test is done as per our requirement.

Temperature should be in between 121 – 124 during sterilization hold time.⁰ ⁰

The steam pressure should be 1.04-1.24 bar during the sterilization hold time.

In this the chemical indicator paper and biological indicator will be used.

The biological indicator is Geobacillus sterothermophillus.

This is a spore producing bacteria which can resist up to 120 C temperature.⁰

Parameters :-Heat up 1 :-105 C⁰Heat up 1 hold :- 02minHeat up 2 :- 108⁰Heat up 2 hold :- 02minHeat up 3 :- 112⁰Heat up 3 hold :- 02 minHeat up 4:-117⁰Heat up 4 hold :- 03minSterile hold temp :- 121.3⁰Control band :- 000.3Sterile hold time :- 20 minSterile hold reset :- 120.5⁰

Sterile hold stop count :- 121⁰Sterile temp over shoot :- 124⁰Jacket control temp :- 121⁰Door open temp :- 96⁰Print interval :- 60 secControl temp :- min for all

Biological indicator for gravity cycle Geobacillus sterothermophillus

Before test After test

HPHV cycle :-

High pressure and high vacuum test cycle is used for the

sterilization of a glass were, accessories etc.,

Temperature should be in between 121 – 124 during ⁰ ⁰sterilization hold time.

The steam pressure should be 1.04-1.24 bar during the

sterilization hold time.

Parameters :-Pre vacuum :- -0.700Bar Pre vacuum hold time :- 02 minPre pressure :- 0.300BarPre pressure hold time :- 02 minHeat up 1 :-107 C⁰Heat up 1 hold :- 02minHeat up 2 :- 112 C⁰Heat up 2 hold :- 02minHeat up 3 :- 115 C⁰Heat up 3 hold :- 02 minHeat up 4:-117 C⁰Heat up 4 hold :- 1minSterile hold temp :- 121.3 C⁰

Control band :- 000.3

Sterile hold time :- 30 minSterile hold reset :- 120.6 C⁰ Sterile hold stop count :- 121 C⁰Sterile temperature over shoot :-

124 C⁰Post vacuum :- -0.700BarPost vacuum hold time :- 02 minDoor open temp :- 90Print interval :- 60 sec

Observations and Results:Vacuum leak test:

Run - I Run - II Run –III

Date 25/05/2016 25/05/2016 25/05/2016

Cycle start time 14;53;50 15:16:37 15:59:19

Vacuum hold start time

15:01:16 15:24:04 16:06:49

Vacuum hold end time

15:11:16 15:34:04 16:16:49

Cycle completion time

15:12:35 15:35:23 16:18;09

Vacuum hold start pressure

-0.723 bar -0.724 bar -0.725 bar

Vacuum hold end pressure

-0.717 -0.717 -0.717

Actual leakage 0.006bar 0.007bar 0.008bar

Bowie Dick test Run –I Run – II Run – III

Date 25/05/2016 25/05/2016 25/05/2016

Cycle start time 16:38:01 18:04:20 19:11:44

Sterile hold start time 17:18:58 18:29:25 19:36:13

Sterile hold end time 17:29:58 18:40:25 19:47:13


17:47:32 19:00:08 20:05:42

No leaks observed Bowie Dick kit turns blue colour to black colour

Steam in process (SIP) test:Run – I Run – II Run - III

Date 27/05/2016 27/05/2016 27/05/2016

Cycle start time 13:36:00 14:29:42 16:14:40

Sterile hold start time

13:45:00 14:39:05 16:24:40

Sterile hold end time

14:00:00 14:54:05 16:39:40


14:03:45 14:57:35 16:42:20

Min. Temp during hold

121.1 121.1 121.1

Max. Temp during hold

124.4 124.5 124.5

Avg. Temp during hold

123.25 123.0 123.8

Min F0 22.79 22.64 27.52

Max F0 26.25 23.41 28.66

Conclusion

After operating and validating the autoclave all the

parameters which are given by USP is satisfied and the

results are also satisfied for all the recipes and all the

cycles what we done for validating the autoclave. So we

concluded that this autoclave is working very efficiently

and sterilizes the media and accessories properly without

any contamination. So now it is used for regular usage of

sterilization.

Thank you