Validation of Dry Heat Sterilizer; perform it best way

Validation of Dry Heat Sterilizer; Purpose

To validate Dry Heat Sterilizer by Biological Indicator & endotoxin Indicator.

Validation of Dry Heat Sterilizer; Scope

This SOP applies for validation of Dry Heat Sterilizer in Sterile Processing area and Microbiology Laboratory at XX Pharmaceuticals Limited.

Definitions/Abbreviation

Validation

Validation is the process to establish documented evidence, which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications & quality attributes.

Biological Indicator

Biological Indicator is a defined preparation of viable spores made from Bacillus stearothermophillus & Bacillus subtilis & has a particular spore count per indicator of not less than 10⁴ and not more than 10⁹ spores which is used to monitor the efficacy of sterilization process.

Endotoxin Indicator

Endotoxin Indicator are designed for monitoring depyrogenation process or validation or which may be measured by comparing the levels of endotoxin before and after a depyrogenation cycle using LAL reagent . USP[United State Pharmacopoeia]  suggests that a depyrogenation cycle should be reduce the endotoxin  by at least 1000 fold ( 3- log reduction ) in endotoxic activity as measured by LAL method.

Endotoxin

It is the component of cell wall of certain bacteria which type of bacteria is known as gram negative bacteria. Endotoxin induce strong immune response and enhance release of cytokine.

[] ATCC: American Type Culture Collection

[] BI: Biological Indicator

[] CSE: Control Endotoxin Standard

[] EI: Endotoxin Indicator

[] λ: Lambda(Lysate Sensitivity)

[] LAL: Limulus Amebocyte Lysate

Responsibilities

The roles and responsibility is as follows

Executive/ Sr. Executive, Microbiology

Preservation of all Indicator, perform Dry Heat Sterilizer validation & report preparation.

Manager, Microbiology

Ensure Sterilizer Validation, documentation and application of sound technical information.

Head of Quality Assurance

Take initiative to approval of this SOP

Procedure:

Instructions

• Biological Indicator is usually non-pathogenic but all microorganisms are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if required).
• CSE[Control Standard Endotoxin] is pyrogenic in humans. Care should be exercised when handling to avoid ingesting it.
• Use caution if handling hot vials. Wear gloves or wait until vials are cool.
• Before transferring BI[Biological Indicator] from Microbiology Laboratory, disinfect whole surface of all apparatus with the help of 70% IPA or any others disinfectants.
• Leave it at designated containers safely.
• Ensure that waste container is tightly capped until autoclaving.

Handling of Biological Indicator & Endotoxin Indicator :

• Use BI[Biological Indicator] for sterilization cycle validation & EI[Endotoxin Indicator] for Depyrogenation cycle validation.
• Read insert package carefully for instruction of use.
• Check expiry date of Biological Indicator (Bacillus subtilis, ATCC-9372) and Endotoxin Indicator.
• Discard it after autoclaving if expiry date is exceeded.
• Observe Certificate of Analysis of all indicators.
• Ensure BI[Biological Indicator] paper strip & EI [Endotoxin Indicator] vial is intact.
• Preserve always BI at 25⁰C and at [2 to 8]⁰C for EI.
• Do not preserve it at the freezer.

Culture Media Sterilization :

• Prepare required amount of CSDM [Casein Soyabean Digest Medium] for the test & distribute [15 to 20] ml of medium in different test tubes.
• Sterilize media at 121⁰C for 15 minutes.
• Preserve those at [2 to 8]⁰C for use.

Validation Frequency

• Perform the validation once in six months.

Exposure Condition:

• Carry out validation as per test schedule.
• Map chamber with most critical area for exposure of BI[Biological indicator] or EI[Endotoxin Indicator].
• Define location & label each BI or EI.
• Place EI at 4 points & BI at 12 points as per chamber mapping, mentioned on Table-1.
• Complete sterilization cycle along with product or empty sterilizer.
• After sterilization, transfer all indicators to Microbiology Laboratory.

BI[Biological Indicator] Assay

• Perform whole analysis under Laminar Air Flow workstation.
• Aseptically open envelopes of BI[Biological Indicator] test strips.
• Place each test strips including negative control and positive control strips in individual tubes containing [15 to 20]ml of TSB.
• Identify all tubes.

Incubation of Biological Indicator

• Incubate the BI strips at [30⁰ to 35]⁰C for 7 days.

Interpretation of Result

Observe BI[Biological Indicator] tube daily for growth. Growth should occur in positive control tube within 48 hours. The turbidity indicates positive growth of tubes. Sterilizer is valid if following criteria are met:

• No growth found in exposed all tubes.
• No growth found in negative control tube.
• Growth found in positive control tube.

Repeat validation if following result is found :

• Growth found in one or more than one exposed tube.
• Growth found in negative control tube.

Sterilizer is invalid if following result are found

• Growth found in one or more than one exposed tube.
• No growth found in negative control tube.
• Growth found in positive control tube.

EI Assay Procedure of LAL Test(Gel Clot Method) :

• Use the LAL reagent sensitivity 0.125 EU/ml for this assay.
• Reconstitute each EI vial with 1.0 ml LAL water including Positive control & vortex at least five minutes and then dilute 1:8 by using LAL water at least duplicate.
• Make a control series (2 λ, λ, λ/2, λ/4) if a new lot of Endotoxin indicator or LAL reagent is used. Otherwise test at λ (Lysate sensitivity) only.
• Carry out LAL test for exposure vials, positive control and negative control as per Standard Operating Procedure for Bacterial Endotoxin Test.

Incubation of Indicator

• Incubate all LAL test tubes including positive & negative control at [37±1]⁰C for [60±1] minutes.

 

Interpretation of Result

• A more exact calculation of endotoxin reduction is made by finding the end-point of the positive control, by ten-fold & then two fold dilution, & subtracting the logarithms of the exposed vial from the logarithms of the positive control.

Use the logarithm of lamda for the endotoxin concentration in exposed vials when there are negative LAL test results for the undiluted solutions.

The sterilizer is valid if the following criteria are met

• No clot formed in all exposed tube
• No clot formed in negative control tube
• Clot formed in positive control tube

Repeat validation if the following result is found

• Clot formed in one or more than one exposed tube
• Clot formed in negative control tube
• Not clot formed in positive control tube

The sterilizer is invalid if the following result is found

• Clot formed in one or more than exposed tube
• No clot formed in negative control tube
• Clot formed in positive control tube

Corrective Action:

• If endotoxin found in anyone exposed vial in the repeat test, inform the test result to concerned department & Engineering Department for corrective action.
• After corrective action, Engineering Department or concerned department shall inform to Microbiology Lab. to perform the validation again.
• Microbiology Section shall perform the complete test.

Report Preparation

Report the validation in Dry Heat Sterilizer Validation Report by Biological Indicator, Annexure-I and Dry Heat Sterilizer Validation Report by Endotoxin Indicator, Annexure-I

Download All Annexure Here:

Annexure I:  Dry Heat Sterilizer Validation Report by Endotoxin Indicator

Annexure II: Dry  Heat Sterilizer Validation Report by Biological Indicator