Microbiological Testing of Packing material
This Document describes (Standard Operating procedure) Sop for Microbiological Testing of Packing material (Inprocess)
Sop for Microbiological Testing of Packing material (Inprocess)
I. Purpose & Scope:
- The purpose of this SOP is to lay down the procedure for Microbiological Testing of Packing material (Inprocess)
- This Standard operating procedure is applicable for Microbiological Testing of Packing material (Inprocess)
II. Responsibilities:
All Quality control personnel’s shall be responsible to follow and implement this SOP i.e. Microbiological Testing of Packing material (Inprocess)
III : Introduction and Procedural Part :
Introduction on Microbiological Testing of Packing material (Inprocess) :
- For sterile block, we use various types of Primary and Secondary packing material such as Rubber bung, Glass Vial and Aluminum seals.
- During filling activity, Rubber bungs and Glass vials are terminally sterilized for packing purpose.
- Rubber bungs are sterilized in Bung processor, Glass vials are depyrogenated in Tunnel vial machine and Aluminum seals are Sanitized by 70% Filtered IPA.
- For Sterility assurance during filling activity, all these materials are needed to check for Bioburden.
Requirement and Transfer of sampling kit :
- For Analysis of Depyrogenated Glass vial and Sterilized Rubber bung, Media required are Soybean casein digest medium (SCDM) and Fluid thioglycollate medium (FTGM).
- For Analysis of Sanitized aluminum seal, Media required are Buffered peptone sodium medium and Dey engley neutralizing agar medium.
- The media used should comply the Sterility and Growth Promotion Test, carried out before, or in parallel, with the test.
- For Sampling purpose, Keep Soybean casein digest medium (SCDM) and Fluid thioglycollate medium (FTGM) tubes, Sterile empty bottle for Sampling of Aluminum seal in SS Sampling kit (SS Drum).
- Transfer the Sampling kit into washing area of sterile block by following respective entry procedure.
- Then sanitize the sampling kit with 70% Filtered IPA and keep it into Dynamic Pass box of Washing room
- Then enter the Filling area of Cephalosporin by following the Entry procedure.
- Take the sampling kit from Dynamic pass box and perform the Sampling.
Examination of Rubber Bung and Glass Vials:
- Perform the Sampling and analysis of rubber bung and glass vial by Direct inoculation method.
- Avoid extrinsic contamination and keep aseptic behavior during Sampling of Rubber bung and Glass vials.
- Sampling of Depyrogenated Glass vials and Sterilized rubber bung should be performed under LAF of Filling Machine.
- For Sampling of Depyrogenated Glass vials, taking approximate 2 to 4 Glass vial in each of Soybean casein digest medium (SCDM) and Fluid thioglycollate medium (FTGM) by Direct inoculation method.
- For Sampling of Sterilized Rubber bung, taking approximate 10 rubber bungs in each of Soybean casein digest medium (SCDM) and Fluid thioglycollate medium (FTGM) by Direct inoculation method.
- After sampling, keep all sampled tubes in Sampling kit and transfer them to Microbiology department for further incubation.
- Incubate Soybean casein digest medium (SCDM) tube at 20 to 25ºC for 14 days, and Fluid thioglycollate medium (FTGM) tube at 30 to 35ºC for 14 days.
- Perform the entry of sample in Inward register of Sterility testing , Observe each tube on daily basis and Record the result.
- Keep Negative control of Soybean casein digest medium (SCDM) and Fluid thioglycollate medium (FTGM) ,incubate at 20 to 25ºC and 30 to 35ºC respectively for 14 days.
- Carry out Positive control on same lot of Fluid Thioglycollate medium using 10-100 cfu/ml viable micro-organisms inoculum of Clostridium sporogenus (ATCC 11437)
- Carry out Positive control on same lot of Soybean casein digest medium using 10-100 cfu/ml viable microorganisms inoculum of Bacillus subtilis (ATCC 6633)
- Incubate the Positive control tubes of soybean casein digest medium at 20o – 25o C and of fluid thioglycollate medium at 30o -35o C for not more than 3 days in case of bacteria & not more than 5 days in case of fungi.
- Record the Observation.
Examination of Sanitized Aluminum seals :
- Aluminum seal analysis should be done by Membrane filtration method.
- For this, we require Empty sterilized bottle, Buffered peptone water and Dey engley neutralizing agar.
- Sampling of Sanitized Aluminum seal should be performed under LAF of Sealing Machine.
- Take randomly approximate 10 Sanitized aluminum seal in Empty sterilized bottle.
- Transfer the bottle to Microbiology department for further analysis.
- Perform the entry of analysis in Inward register of Packing material ,refer Annexure I.
- Further analysis should be performed Under LAF of MLT Room .
- Transfer the approximate 100ml of Sterile Buffered Peptone water solution in Sample bottle containing aluminum seals.
- Then Vigorously Shake the bottle for 1 min , then filtered through Sterilized filtration assembly (0.45 µ membrane filter paper, size 47mm).
- After filtration, lift the membrane filter with sterilized forcep and place it on the surface of Dey engley neutralizing agar (DENA) Plate. Avoiding Air bubble entrappment under membrane filter. Incubate the plate in inverted position.
- Perform the Dual temperature incubation condition for Incubation of Plates.
- At first Incubate the DENA Plates at 20-25°C for 72 hours in inverted position, for determination of TFC. then incubate the same plates in the inverted position, at 30-35°C for 48 hours for determination of TBC.
- Count the number of colony forming unit (CFU) observed on the plate after 5 days. Total Viable Aerobic Count (TVAC) is the summation of the TBC & TFC. The count is expressed as CFU / Record the results in Annexure- II.
- Positive control: For positive control, Refer the media fertility test performed during Media preparation.
- Negative Control: Keep one plate of the same lot of media used for testing, as negative control and incubate at respective temperatures along with test.
Limit and Frequency :
| Activity | Maximum Limit | Frequency |
| Total Bacterial Count | 50 cfu/plate | During Filling Activity of Each batch |
| Total Fungal Count | 02 cfu/plate |
Action to be taken if microbial count Exceed the limit :
- On Observation of Out of limit results in the samples, the Microbiologist shall inform to Head of Quality Control and Quality assurance.
- After Review the results, Start the Out of Specification investigation as per specified SOP for further investigation.
IV: Annexure for Microbiological Testing of Packing material (Inprocess) :
Annexure I: Inward Register of Bioburden analysis of Primary packing material
Annexure II : Bioburden Report of Inprocess Sample of Primary Packing material
Annexure I
Inward Register of Bioburden analysis of Primary packing material
| Date | Name Of Material | Batch
No. |
A.R.No. | Enumeration count (cfu/plate) | Completed on | Analyzed By/On | Checked By/On | |
| Total Bacterial count | Total fungal count | |||||||
ANNEXURE II
Bioburden Report of INPROCESS SAMPLE OF Primary Packing material
| Name of Material | Type of Material | ||
| Batch No. | A. R. Number | ||
| Date of Sampling | Date of Analysis | ||
| Media Lot Number | Analysis Done by | ||
| Incubation Condition | 20-25°C For 72 hours | Incubator ID No | |
| Incubation Condition | 30-35°C For 48 hours | Incubator ID No | |
| Date of Completion | Observed by |
- A) Sample Preparation:
Add Seals in ml of (Solution A )
Filter the Solution A, through Sterilized filtration assembly.
- B) Total Enumeration Count :
| Name of Material | Total Viable Aerobic Count (cfu/Plate) | Maximum Limit
(cfu/plate) |
||
| Total Bacterial Count | Total Fungal Count | Total Bacterial Count | Total Fungal Count | |
| 50
|
02
|
|||
Negative Control : Growth observed / not observed.
Conclusion: The Bioburden of Sample found Satisfactory/Not Satisfactory.
Observed By: Checked By:
Date: Date:
