MICLAB-035 Aseptic Media Filling and Microbiology Integrity Leak (Soup) Testing Procedure

All care must be taken when preparing media fills with HOT water.  Rubber gloves are to be worn when using HOT water.

Safety glasses and gloves are to be worn when using 70% IPA.

1. Introduction

The procedures set out in this SOP should be carried out TWICE every year on all the filling equipment, at defined 6 monthly intervals on each production shift. Three successful media fills should be conducted as part of the validation/ commissioning of any new piece of equipment or after any significant re‑design of existing equipment.  (See MICLAB 040) To demonstrate container integrity in a new process or after any significant change that may affect the integrity, Microbiological Integrity Leak test (“Soup” test) may be required, (Form 665).

2. Procedure

2.1.         A representative from the Microbiology Laboratory team is to attend the scheduled meeting the week prior to a media run being performed on a process. At this meeting they will outline the purpose of the media run, the type and size of product container to be used and hence the volume of medium required (determining a minimum number of units to be filled with sufficient medium), the volume required is dependent on the process to be evaluated.  They will go over the Intervention matrix to predetermine what routine interventions and non-routine interventions need to be conducted during the media run.

Refer to Appendix 1 for Intervention matrix.

2.2.         The Microbiology Laboratory staff will arrange for sufficient dehydrated Tryptone Soy Broth medium (as required).

2.3.         The Microbiology Laboratory staffs are to initiate filling in Form 670 for each batch of media which then moves with the process flow of the media run and is returned from production when completed and is to be stored in the “In-Process Media Run” file.  The documentation is to be completed and if there is no data to be entered then N/A must be written in the space provided.

The Process teams should their checklist for documentation control and all documentation is to follow standard procedures and is to be sent to the QA Department. Form 670 is sent to the Microbiology Laboratory with the media run, along with any media waste and rejected media filled containers.

2.4.         Level of fill for media filled containers and “Soup” tests, should be not more than 80% of the volume of the container where possible, however some processes need correct volumes fill for the inspection process.

2.4.1.     Aseptic filling – containers to be at least 2/3 filled, to correct fill volume.

2.4.2.     Terminally sterilized – containers to be at least 2/3 filled, to correct fill volume.

Note     Sterile medium will be transferred to the filling machine as follows:

Method (a)        Filter sterilization of the medium from a Dispensing Pressure vessel directly to the filling machine.

Method (b)        Filter sterilization of the medium from a Dispensing Pressure vessel into a sterile 50L or 100L glass storage vessel and then transfer this vessel to the sterile filling area for filling into containers.

When sterile filtering for any of the methods, install a pre-filter before the primary filter to prevent it from clogging.  If the pre-filter clogs during the run it can be changed to a new filter, as long as the primary filter stays in place and sterility is not broken. This pre-filter cannot withstand steaming in-place, so it is to be installed just before filling is to start.

3. Media Preparation

Prepare the required volume of Tryptone Soy Broth by dissolving the correct amount of dehydrated powder in hot (80°C) Distilled Water.  Simmer the medium on the hot plate and reconstitute if necessary while transferring to either a 20 or 40 liter Dispensing Pressure Vessel.

Notes   1.    The medium may be heated in a double or triple strength form and then diluted with the additional water required when being transferred to either a 20 or 40 liter Dispensing Pressure vessel.

Filter sterilization of the medium must be carried out within 4 hours of preparation of the medium.  The “4 hours” may be extended if the media is sterile-filtered into a pre-sterilized vessel.

The start time of the media run can be taken from the point when solution is sent down the line to start the wetting of the Sterilization filter to conduct the Pre-Integrity test.

4. The Aseptic Media Fill

The Aseptic Media Fill must simulate as closely as possible a normal production run.  It should cover worst case conditions or activities (however not to the point of failure), e.g. filling rates, number and activity level of Operators, temperature and should be run on different shifts where possible to cover all operators, etc.  The duration of the media run should be at least 4 hours or half a production shift to allow for all routine interventions (see Appendix 1).

4.1.         Environmental monitoring

During the media run environmental monitoring is to be conducted including the following:

Settle plates (the number of settle plates will depend on the machine)

Air sampling 1000L under the machine laminar flow and 200L in the filling room.

Surface plates of the floor and machine.

Personnel Monitoring for Sterile operators.

Note: After monitoring has taken place, all plates are to be sealed using tape and delivered to the Microlab Sampling cabinet and logged into the appropriate logbook.

4.2.         The filled units received by the Microbiology Laboratory are to be placed into the 30°C (±1.5) Hot room.  The Microbiology Laboratory personnel are to inspect the containers for evidence of microbial growth after 7 and 15 days incubation.  During the 7day inspection, shake the contents of the container to ensure that the media has come in contact with all internal surfaces of the container.  Store back into either shippers or buckets in a different orientation to the first incubation period.  Record all additional information onto the “Aseptic Media Fill Information Sheet” (Form670) and in the media fill Manufacturing Instruction sheets, which were initiated at the manufacture of the medium used for the media run.

4.3.         If any containers show evidence of microbial growth raise a DR.  Inform Microbiology Manager, Production Manager and review the possibility of off line dye testing to confirm container integral. Then open the container and streak (for individual colonies) the contaminated broth onto a Nutrient Agar plate and incubate at 30°C (±1.5°C) for 24 hours.

4.4.         If the contaminant/s is a bacteria or yeast, identify to Genus level and if possible species level (MICLAB 070).  Record the details of morphological appearance Gram stain reaction and identity (if determined) on Form 670 and in the Media Fill manufacturing instruction sheets.

4.5.         After the required incubation time (15days) the medium must be checked for its ability to promote the growth of low levels of microorganisms. Under Laminar Flow, aseptically pool the medium from several containers into at least 6 sterile 100mL bottles or if possible, e.g. with vials, directly inoculate the media-filled containers with microorganisms as detailed below.

Inoculate 1/3 of these bottles with Staphylococcus aureus at levels of ideally 10-20, but definitely less than 100 viable organisms and incubate at 30°C (±1.5°C).

Inoculate a further 1/3 of the bottles with Candida albicans at a similar level and incubate at 30°C (±1.5°C).  Inoculate the final 1/3 with Bacillus subtilis at a similar level of viable spores and incubate at 30°C (±1.5°C).

Note     At least once per year, for a media run conducted on each piece of filling equipment, substitute an environmental organism isolated from the Sterile filling area for one of the above micro-organism.

Examine these bottles daily for evidence of microbial growth.

Growth should be evident in all the bottles within 48 hours incubation.  The inoculum can be obtained by appropriate dilution of the stock suspensions of the organisms held in the Microbiology Laboratory refrigerator and the level used checked by plate count.  See MICLAB 090.

Record results of the medium’s ability to promote the growth of microorganisms on Form 670 and also in the Media Fill manufacturing instruction sheets.

4.6.         Acceptance Criteria– the target is zero positives

The target should be zero growth but a contamination rate of less than 0.1% with 95% confidence level is acceptable.

Reoccurring incidents of contaminated units in aseptic media fills for an individual line, regardless of the acceptance criteria, would be a signal of an adverse trend on the aseptic processing line. This would lead to a DR being raised and an investigation started to look for root cause, corrective action and possible revalidation.

Reject levels should be consistent with normal production reject levels.

Note: There may be a requirement as part of registered details for media runs to be conducted on both terminally sterilized process or non-sterile manufacturing process.  Acceptance criteria will differ from aseptic process.  If there is one positive unit found in the process media fill, a DR will be raised and we will undertake an investigation and determine appropriate corrective actions.

4.6.1.     The medium must be shown to be able to promote the growth of low levels of microorganisms, etc.

If the medium fails to promote the growth of low levels of microorganisms and/or the limits are exceeded, inform the Microbiology Manager, Quality Assurance Manager and the Area Manager immediately, a DR is to be raised and an investigation conducted.  The Aseptic Media fill is invalid and must be repeated as soon as practical.

4.6.2.     Micro. Lab. personnel are to transfer the remaining media‑filled containers to the Security Rejects Bin in accordance with MICLAB 020.

4.6.3.     A Validation Report is to be written by a Microbiology staff and approved by the Microbiology Manager. The report is to detail the procedure, interventions conducted both, routine and non-routine, results of the Media Fill run and is to be kept in the Aseptic Media Fill Validations file in the Micro. Lab.

As part of the report, Micro. Lab. will receive back all documentation sent to the QA Department concerning the media run, along with media run checklist.

When writing the report micro lab staff should review the for DRs related to the batch for the media run.

4.6.4.     The report is to be issued within 1 month of the completion of the test.

5. Microbiological Integrity Leak Test (“Soup” Test)

5.1.         The test is to be conducted as per the Protocol – (use Form 665). The Protocol will change according to the particular circumstance.

6. Appendix 1 – Intervention Matrix:

Minimum requirements to be conducted annually are as outlined below.

7. Summary of Changes