SOP for Procedure for Bioburden, BET and LPC of Primary Packaging Materials

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1.0  PURPOSE

       To lay down the Procedure for Bio-burden, BET and LPC of primary packaging materials.

2.0  SCOPE

       This is applicable to Microbiology lab

3.0  RESPONSIBILITY

       Microbiologist

4.0  ACCOUNABILITY

       Head of Department

5.0  PROCEDURE

5.1  Bio-burden:

5.1.1  Collect 10 units each of vials, rubber stopper, and seals and bottle randomly. Collect 10 gm sample in case of aluminium foil and PVC sheet as per SOP for sampling packing material.

5.1.2  Use clean and sterilized sampling devices to avoid external contamination.

5.1.3  Bring sample to microbiology lab for testing.

5.1.4  Carry out test under LAF.

5.1.5  For Vials and Bottle: Fill approximately half of the total volume of vials and bottles aseptically with Sterile saline. Shake vials and bottles in such a way that internal surface of vial and bottle are to be rinsed properly.

5.1.6  Filter aseptically whole content of each vial through sterilized filtration assembly (0.45μ membrane filter) and rinse with 100 ml of sterilized 0.1% peptone water.

5.1.7  Lift membrane filter with sterilized forceps and place it on pre-incubated soybean casein digest agar plate avoiding air bubble entrapped under filter paper.

5.1.8  Incubate plate at 20-25⁰C for 72 hrs. followed by 30-35⁰C for 48 hrs.

5.1.9  For Rubber Stopper and Seals: Under aseptic condition dip sample of seal and rubber stopper in each of 100 ml of sterile saline, shake it properly and filter through sterilized filtration assembly (0.45μ membrane filter) and rinse with 100 ml of sterilized 0.1% peptone water.

5.1.10  Lift membrane filter with sterilized forceps and place it on pre-incubated soybean casein digest agar plate avoiding air bubble entrapped under filter paper.

5.1.11  Incubate plate at 20-25⁰C for 72 hrs. followed by 30-35⁰C for 48 hrs.

5.1.12  For Aluminium Foil and PVC Foil: Prepare sterilized swab in saline and Take aseptically swab of 5X5cm area from the internal surface of aluminium foil and PVC sheet with the help of sterilized 5X5cm template in such a way total 5X5 cm area is to be covered properly.

5.1.13  Dip swab stick in saline tube and vortex it properly and filter whole content of saline through sterilized filtration assembly (0.45μ membrane filter) and rinse membrane filter with 100 ml of sterilized 0.1% peptone water.

5.1.14  Lift membrane filter with sterilized forceps and place it on pre-incubated soybean casein digest agar plate avoiding air bubble entrapped under filter paper.

5.1.15  Incubate plate at 20-25⁰C for 72 hrs. followed by 30-35⁰C for 48 hrs.

5.1.16  After incubation take observation and record the result.

5.2  Bacterial Endotoxin Test (BET):

5.2.1  Collect two units each of vials and fill with Lal Reagent Water (LRW) in suitable volume and vortex it for 5-10 min. Pool sample in separate pyrogen free tube.

5.2.2  In case of rubber stopper collect two stopper and dip in 2 ml of LRW and vortex it for 5-10 min.

5.2.3  Performed test as per SOP.

5.3  Liquid Particle Count (LPC):

5.3.1  For vials: Collect 15 vials for 20 ml capacity and two vial in case of capacity more than 20 ml under LAF as per SOP.

5.3.2  Fill required vials with blank water.

5.3.3  Take aliquot in 100 ml particle free beaker for testing.

5.3.4  Check the particle against blank as per SOP.

5.3.5  For rubber stopper: Take wide mouth capped bottle with particle free aliquot (100ml) for sampling.

5.3.6  Collect 20 rubber stoppers under LAF as per SOP.

5.3.7  Put rubber stopper directly into the bottle.

5.3.8  Bring rubber stoppers to the lab separate the aliquot under LAF.

5.3.9  Check the particle against blank as per SOP.

5.4  Test frequency:  

Initial three consignments of every new vendor and after every three month.

6.0  ABBREVIATIONS

6.1  SOP - Standard operating procedure

6.2  LAF - Laminar air flow bench

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