MEDIA FILL PROCESS AND ITS VALIDATION

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What is Media Fill ? The Media fill or Broth fill technique is one in which a liquid

microbiological nutrient growth medium is prepared and filled in a simulation of normal manufacturing operation.

The microbiological growth medium such as Soybean Casein Digest Medium (SCDM)is processed and handled in a manner which simulates “normal” manufacturing process with same exposure and possible contamination.

The final container is then incubated and checked for turbidity which indicate the microbial contamination.

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•Why the validation of aseptic process is required by pharmaceutical regulations?A “sterile product” is defined as “free of viable organisms”As it is not practical examine every unit for confirmation of sterility, all efforts are made to minimise the risk of contamination (finishing, HVAC, pressure differentials, cleaning procedure, monitoring programme)Despite of many measures taken, contamination is an ever-present danger because aseptic processing is a process being operated in a controlled –but not sterile- environment and sample numbers are too small; so that only gross contamination is likely to be detected

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• Although media fills must duplicate aseptic manufacturing conditions, it is not possible for them to be conducted in exactly the same way as the manufacture of a production batch of a pharmaceutical product.

• In aseptic processing, the greatest risk comes from the personnel working in the clean room: the operators have to participate in media fills.

• Environmental monitoring activities are required during aseptic filling operations.

• It is usual to include the “worst case” conditions that can occur in production runs.

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Aseptic process:

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washing machinedepirogenating tunnel

filling machine

stoppering machinecapping machine

Hands-on: Aseptic liquid filling line

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Media Fill Protocol• Number and frequency of runs• Medium culture (to replace the product)• Number of units filled • Container (vial) size• Fill volume• Line speed (or filling speed)• Duration of fill• Operators shifts• Monitoring activities• Interventions –both routine and non-routine• Incubation method• Acceptance criteria

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Number and frequency of runs

In start up simulation at least three consecutive separate

successful runs should be performed (it is recommended they are

performed in different days).

For on-going simulation, a routine semi-annual qualification is

recommended (one run)

Extraordinary media fill should be performed after all changes to

a product or line changes evaluated as a potential danger for the

aseptic process.03/05/23 9

Medium culture

The medium needs to support the growth of a wide variety of

microorganisms, including aerobic bacteria, yeasts and moulds

(non-selective medium).

For aerobic conditions: Soybean Casein Digest Medium (SCD)

also known as tryptone soya broth (TSB).

For anaerobic conditions: usually in a nitrogen environment fluid

thioglycollate medium (FTM).

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Medium culture

The media have to support the growth of microorganisms (growth

promotion test). The organisms to be tested are stated by

pharmacopoeia.

Generally at the end of incubation period, some vials (taken from

the beginning, at half and at the end of the process) are inoculated

with < 100 CFU and incubated for 3 days (bacteria) and 5 days

(yeast and mould).

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Number of units filled

Number of units filled should reflect the real batch size.

It is allowed to fill a lowest number of units provided that the

number of units filled is sufficient to reflect the effect of potential

operator fatigue and adequately represents the maximum number of

interventions.

Some regulations suggest the number of units to be filled in

consideration of batch production size.

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Container size

The extremes of size containers should be considered.

The largest container (often filled at the lowest speed because of

its large fill volume) often has the large opening , so the potential

for microbial entry from the environment should be the greatest for

that size.

The smallest container (often filled at the highest speed for its

lower fill volume) represents the greatest handling difficulty; the

smaller container are more fragile and less stable and be more

subjected to breakage and jamming in the equipment.03/05/23 13

Container size

In the initial qualification two runs might be performed using the

largest container and the third run using the smallest container

In routine evaluation of the line, any container should be included

in the validation program

Clear containers should be used as a substitute for amber

containers to allow visual detection of microbial growth

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Fill volume

The volume of media filled into the containers need not the

routine fill volume.

It should be sufficient to contact the container-closure seal

surfaces (when the unit is inverted and swirled) and sufficient to

allow visual detection of microbial growth post incubation.

Smaller containers should not be over-filled as sufficient air

must be available in the container headspace to support the growth

of aerobic organisms (generally 25% of volume is not filled).

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Line (or filling) speed

The media fill should address the range of line speeds employed during production. Sometimes more than one line speed should be evaluated.The speed chosen for each batch during simulation should be justified.Use of high line speed is justified for manufacturing processes characterized by frequent interventions or a significant degree of manual manipulation. Use of low speed is justified for manufacturing processes characterized by prolonged exposure of sterile components in the aseptic area.

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Duration of fillIn general media fills should be long enough to include all of the required interventions and stoppage and should reflect the potential operator fatigue: a typical media fill might be at least 3-4 hours long. Ideally a media fill should use more units than are in the product being simulated (for all batches up to 5000 units). For very large batches or long campaigns, some blank units (either empty or water filled) are used to maintain operating conditions during the simulation: this technique can be used to validate processes that may run for several days in order to validate the full length of the longest approved campaign

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Operators shifts

Each operator performing aseptic processes are requested to participate in media fill.Set-up and line operators should be part of not less than one process simulation per year. Operators such as line mechanics and environmental samplers should be managed in a similar manner.A maximum number of personnel present in the aseptic processing room should be established.When a firm operates on multiple shifts, the second and third shift should be included in the media fill programme.In case of manual operations (filling), each line operator should participate into all three initial validation runs and at least one run in re-validation (every six months)

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Environmental monitoring activitiesThere are regulatory and pharmacopoeia references that states the microbial conditions.Air sampling using either active and passive sampling methods should be performed during the execution of the process. Surface sampling is best performed at the end of aseptic process. Also personnel should be monitoredMicrobiological monitoring (air, surfaces, personnel) and particle monitoring should be performed during media fill employing the same procedure in force Sometimes the number of sampling locations might be increased respect the routine procedure

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Interventions –both routine and non-routine

Media fill records should document all interventions performed and the number of units removed.Routine interventions: aseptic line set-up in which sterilised parts are removed from protective materials and installed is a potential danger; it is common to identify the first containers filled as they may be more indicative of potential problem with aseptic assembly.Other routine interventions: stoppers bowl feeding, remove fallen vials, remove jam stoppers, operators breaks, gloves change, environmental monitoring.Non routine interventions (occur randomly): glass breakage, change / reset of filling needles, interventions on weight adjustments, sensor failure, rail adjustments.

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Incubation methodsAny filled units should be inspected prior to incubation; any defects that compromise the container closure or non-integral units are rejected and documented.Divergence in industry practice: incubation is performed for 14 days at 20-35°C (+/- 2,5°C): it is performed for 7 days at 20-25°C and further 7 days at 30-35°C; it is performed for 7 days at 30-35°C and then move the filled containers to 20-25°CThe lack of agreement suggest that the selection of incubation conditions employed.Units are incubated in an inverted position for the first half of the incubation period and then returned to an upright position for the remainder.

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Acceptance criteria

The target should be zero growth but a contamination rate less than 0,10% with 95% confidence level is acceptable (approx. 1 contaminated unit in 5000 filled units).FDA and PDA agree that the target should be zero contaminated units regardless of size of runIt is important to note that “invalidation” of a media fill run should be a rare occurrenceEach failure should be investigated

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Filled units per run

Contaminated units permitted (action level)

3000 04750 16300 27750 39150 4

10510 511840 613150 714430 815710 916960 10

The table indicates the maximum permitted number of contaminated units per various Media Fill “run sizes” to indicate a 0,10% contamination limit with a 95% confidence level

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Contamination

• The root cause of a failure (contamination), or at least the most probable one, must be identified

• It is important to be able to isolate and identify (to species level) the microorganisms

• An appropriate corrective action / preventive action plan must be implemented

• The impact of the failure on product lots already released (if any) must be evaluated

• After the corrective actions have been implemented, a new media fill study is performed to confirm their efficacy

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CR decreases during filling

Con. spike during filling

Con. spike followed by an increase

Isolated events (few contaminated units per

run)

CR increasing during filling

Validation

OBJECTIVE The Objective of validation protocol is to establish

documented evidence that the process employed for aseptic processing of Parenterals liquid/Ophthalmic solution will produce the desired results consistently, within the specified acceptance limits, when performed as per the latest Standard Operating Procedures.

SCOPEThe Validation protocol describes the procedure for the

total Process Simulation (Media Fill).

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RESPONSIBILITIESSr. no . Responsibility Name of the

department

1 Preparation of Protocol QC

2 Provision of qualified personnel to assist in the protocol preparation and execution

QC, QA, Production and Maintenance

3 Verification of Protocol QC and Production

4 Approval of protocol QA

5 Final determination of System Acceptability

QA

6 Review and assembling of data into a final report

QA

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PRE-REQUISITES

• Approved Soybean casein digest broth

• Environmental Monitoring of manufacturing areas by Plate Exposure, Air sampling and surface monitoring procedures and its SOP’s.

• Qualified and validated manufacturing equipments, system facility (i.e. HVAC, water, compressed gases) CIP and SIP procedures.

• Trained operating personnel’s.

• Approved BMR for media fill trial.03/05/23 29

Check and ensure that-The equipment and system facility is validated. The HVAC system, compressed air, CIP and SIP

procedures are qualified.All operations, cleaning/sanitization procedures are

established and operating personnel are trained.Media used for Process Simulation is passed for GPT.The WFI used for preparation of batch is complied to

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IDENTIFICATION OF CRITICAL CONTROL MONITORING PARAMETER

VALIDATION PROCEDURE Main steps for the Validation of the integrated line by

media fill test

1. Cleaning of the line

2. Dispensing of Soybean Casein Digest Medium for 150 L batch size

3. Batch Preparation 150 L

4. Filling And Sealing

5. Incubation and Examination of Media Filled Units

6.  Interpretation of Results

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VALIDATION PROCEDURE

1. Cleaning of the SVP line Carry out cleaning of SVP mixing tank and holding tank along with product

line and bottle pack machine as per respective SOP for CIP. At the end of cleaning, collect last rinses sample from sampling point and send

to QC department with written information for testing of previous product traces.

  After getting approval report from QC, affix status label on the tank “READY FOR STERILIZATION”.

Immediately carry out the sterilization of SVP holding tank along with final filter and product line of bottle pack machine as per respective SOP.

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2. Dispensing of Soybean Casein Digest Medium for 150 L batch size

Enter to dispensing room as per SOP for entry exit procedure to dispensing area.

Check for the clearance of the area from any unwanted materials. Check for the cleanliness of the area, LAF, weighing pan as per checklist. Put “ON” the reverse LAF unit 15 minutes before dispensing of material.

Check the availability of clean containers, pressure differentials, and temperature & humidity should be not more than 250C and 45 to 60% RH respectively.

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Calibrate the balance as per SOP of Balance Calibration.

Take the Approved Soybean Casein Digest Medium in pre-dispensing room, place on SS pallet and check the label of container for correctness and Approval of material.

Transfer the material to Dispensing room, place the empty clean container on the balance and record the tare weight. Press “ZERO” of the balance and weigh the required quantity of material, note the weighed material and then remove the container from balance and press Zero.

Close the dispensed material, affix the weighing tag and transfer the material in dispensed material storage room.

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After dispensing, put “OFF” the balance and LAF. Clean the surrounding area, balance and spray with 70% IPA solution.

Reseal the original container and shift to their original place.

• Batch Preparation 150 L: Ensure that the area and product line is clean and free from

the traces of previous product. Recheck gross weight of Soybean Casein Digest Medium

(SCDM) to be used for manufacturing and ensure that they match as per entries made in the BMR weighing sheet.

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Check the status board affixed on the tank “READY FOR USE”, also verify the records and ensure that the bottom outlet valve of the mixing tank is closed.

Send the entry point sample of WFI from the user point to QC department for testing along with BMR.

On approval of WFI sample from QC department, affix a status board on the Mixing tank “UNDER MANUFCTURING” with Product name and B. No.

Collect approx 50 L water for injection at 80 to 850C in a manufacturing tank fitted with stirrer.

Start the stirrer and add SCDM through the mainhole of the tank.

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Continue stirrer for complete dissolution of ingredients.

Stop the stirrer. Make up the volume to the 150 L with water for

injection. Start the stirring for complete dissolution of SCDM

and homogeneous bulk solution (generally required 10 minutes).

Collect sample of bulk solution in a sterile sampling bottle and send it to QC for testing of color clarity, pH and bioburden along with bulk intimation slip.

After getting clearance of bulk analysis from Quality Control, start the filtration from mixing tank to Holding tank of line with the help of pump.

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After getting clearance of bulk analysis from Quality Control, start the filtration from mixing tank to Holding tank of line with the help of pump.

Perform the bubble point test of the final filter after holding tank as per SOP of Bubble point test.

4.Filling And Sealing: Start the filtration from holding tank to FFS machine using

pump. Drain one buffer tank approx 1.3 liters of bulk solution from

filling nozzle to eliminate any possibility of dilution of bulk by condensates in product line of the machine post SIP.

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Check online cartridge filter integrity test as per its respective SOP.

Start Machine line and discard initial 15 shots. Collect first cassette of vials from next shot and send the

sample with written information to QC for testing. Arrange the out coming cassettes of vials sequentially in

vacuum chamber tray and verify the results of testing from QC department.

Now start the filling and sealing continuously as per SOP for Filling and sealing.

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Collect the filled and sealed containers coming out of the filling area in plastic crates.

During filling operation keep the filled ampoules separately for each breakdown, shift change, power breakdown, stoppage etc and assign lot number.

Arrange the cassettes of vials lot wise in SS trays vertically in vacuum leak testing chamber tray and carry out the leak testing at 650 – 720 mm Hg for 30 minutes. Do not use the leak vials for further media fill study.

After leak test, transfer the goods vials in the clean plastic crates horizontally in cassette from one above the other, lot wise separately

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5. Incubation and examination of filled units:

a. Incubate all media filled units in normal position after leak test at of 20 to 250C for 7 days. Incubation temperature should be maintained within 22.5 ± 2.50C .

b. After completion of 7 days Incubation at 20 to 250C, invert the units and incubate them at 30-350C for next 7 days. Incubation temperature should be maintained within 32.5±2.50C .

c. Each media filled unit should be examined by trained Microbiologist after 3rd day, 7th day, 10th day and 14th day.

d. All suspect units identified during the observation should be brought to the immediate attention of the QC Microbiologist.

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Interpretation of Results:When filling fewer than 5,000 units, no contaminated units should be detected. 

oWhen filling 5,000 to 10,000 units : One contaminated unit should result in an investigation,

including consideration of a repeat media fill Two contaminated units are considered cause for

revalidation, following investigation.

oWhen filling more than 10,000 units : One contaminated unit should result in an investigation;Two contaminated units are considered cause for

revalidation, following investigation. 03/05/23 42

References1. R. A. Nash and A. H. Wachter “Pharmaceutical Process

validation”; Third edition2. Agallo James, Carleton J. Fredric “Validation of

Pharmaceutical Processes”; Third edition 3. P.P.Sharma “How to practice GMP’s” ;Third edition.4. USP <797> ‘media fill testing’ / <71> ‘growth promotion

test’5. FDA “guidance for industry, sterile drug products produced

by aseptic processing – cGMP” 6. PIC/S PI 007-2 “recommendations on the validation of

aseptic process

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