Validation Protocol for Hold Time Study of Sterilized Glassware and Accessories : Pharmaceutical Guidelines

Validation Protocol for Hold Time Study of Sterilized Glassware and Accessories

Learn how to validate the sterilized glassware and other analysis accessories to hold for a period of 5 days under LAF.


       Objective of this protocol is to provide documented evidence through the scientific data to establish and verify the microbial presence on sterilized glassware and accessories upon holding up to specified time after sterilization by using the microbial detection method.

2.0  SCOPE

       The scope of this protocol is to evaluate the hold time of sterilized glassware and accessories after sterilization in auto clave.

3.0  Reference Document

4.0  Responsibility

4.1  Quality Assurance
       Review and approval of protocol
4.2  Quality Control
       Preparation, review and execution of protocol


       Head QA & QC

6.0  Procedure

6.1  Pre-Requisite

Environmental conditions should be maintained within the established limit.

6.2  Procedure For Hold Time Study

6.2.1  Perform the sterilization of the following material Screw capped bottles, Miliflex funnels, SS frits and tips as per the SOP for sterilization.
6.2.2  Store the sterilized material into the laminar air flow.
6.2.3  Collect the samples at regular intervals of 0 hr, 24 hr, 48 hr, 72 hr, 96 hr, and 120 hr and perform the sterility test.
6.2.4  Collect the bottles at the interval as mentioned above and pour SCD medium into the bottle aseptically.
6.2.5  Collect the Miliflex cup at the interval as mentioned above and filtration 100 ml sterile water as per SOP; aseptically transfer membrane into SCD medium.
6.2.6  Collect the SS frits at the interval as mentioned above and transfer aseptically SS frit into 100 ml SCD medium.
6.2.7  Collect the tips at the interval as mentioned above and transfers aseptically tip into 100 ml SCD medium.
6.2.8  Incubate the samples at the specified temperature for 14 days (20-25°C for 7 days and 30-35°C for 7 days).

6.3  Acceptance Criteria

This study is carried out to establish the hold time of sterilized glassware and accessories after sterilization but no growth should be observed.

7.0  Conclusion

After complete evaluation of the hold time study for sterile glassware and accessories used in microbiology lab a final hold time study summary report shall be prepared which should essentially contain discussion and conclusion which clearly determine the hold time period for sterile glass ware and accessories.

8.0  Abbreviations

8.1  SOP – Standard operating procedure
8.2  SCD – Soybean Casein Digest Medium
8.3  °C – Degree centigrade
8.4  SS – Stainless steel

Date of Test:                            Sample:                                               Date of Release:
Media lot No.                          Media Prepared on:                             Media Used Before:
Incubator ID
Start date
End date

Incubation: 20-25 °C for 7 days followed by 30-35°C for next 7 days.

S. No.
Incubation Condition
Observed by
SS frit
-Ve control

20-25°C for 7days
30-35°C for 7days
+ve control:                                                                                   –ve control:
Result:  The sample complies / doesn't comply for sterility.

Done by:                                                                                              Checked by:
Date      :                                                                                              Date           :

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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