Aseptic Filling Process Validation Media Fill Trial

Microbiology SOP,S

In this SOP we will explain the Aseptic Filling Process Validation Media Fill Trial.


To ensure that aseptic manufacturing, filling techniques or practices being used are capable of producing the sterile products.


It is applicable to the injectable area of the Production Department.


Quality Assurance officer

Section Production Officer


Production Manager

QC Manager


4.1 General Conditions

4.1.1 Follow the procedure as closely as possible according to the routine aseptic manufacturing process and all critical subsequent manufacturing steps.

4.1.2 All equipment should remain the same wherever practicable as for the routine process.

4.1.3 Each operator who normally participates in the aseptic fill products should participate in the process simulation.

4.1.4 The process simulation should represent a ‘’worst case’’ situation and include all manipulations and inventions likely to be represented during a shift.

4.1.5 The fill volume of the containers should be sufficient to enable contact of all the container-closure seal surfaces when the container is inverted and also sufficient to allow the detection of microbial growth.

4.1.6 At least 3000 units should be filled in process simulation. For operations with a production size of less than 3000, the number of media-filled units should at least equal the maximum batch size made on the processing line.

4.2 Media Selection and Preparation

4.2.1 The criteria for the selection of growth medium includes:



Ability to support the growth of a wide range of microorganisms (Low Selectivity)

4.2.2 The media should be tested for Growth Promotion as per SAP # — before use.

4.2.3 For process simulation of liquid injectable, use Soybean casein digest broth, however, Fluid Thioglycollate Medium can also be used for the detection of anaerobic organisms, especially where filled nitrogen purge is used.

4.2.4 For Process Simulation of Sterile Dry Powder injectable, use an inert material i.e. polyethylene Glycol (PEG) 8000 in powder form along with a growth medium i.e. Soybean Casein digest broth.

4.2.5 Prepare the Soybean Casein digest broth according to the manufacturer’s instruction in a compounding vessel, dissolve completely and sterilize it by autoclaving at 121ºC & 15lbs for 30 minutes. Allow the medium to cool at room temperature.

4.2.6 PEG should be sterilized. Perform the sterility test of irritated PEG 8000 by membrane filtration.

4.2.7 Handle the growth medium and PEG 8000 in a manner similar to the production process being simulated.

4.3 Sterilization of Equipment, Containers, and closures

4.3.1 Sterilize the equipment or machine parts and closures or rubber stoppers by steam sterilization and monitor the sterilization by using an indicator.

4.3.2 Determine the Bioburden of washed vials.

4.3.3 Sterilize the containers (washed ampoules and vials) by dry heat sterilization and monitor the sterilization.

4.3.4 Determine the pyroburden of sterilized rubber stoppers, ampoules, and vials

4.4 Filling Operations

4.4.1 The filling machine is operated at the present fill rate for the container size utilized, as well as the fastest speed (handling difficulty) and the slowest speed

4.4.2 All routine activities which take place on the filling line should be a part of the process simulation.

4.4.3 Manipulations of routine process must be followed to represent the worst case for example malfunctioning of HVAC system or electricity for more than 10 minutes, increase in a number of operators, etc.

4.4.4 Process Simulation of Liquid injectable

Filter the media under the sterile nitrogen gas pressure using the fiberglass pre-filter and 0.45µm cartridge filter or 0.45µm membrane filters

Before filtration of some amount of media, add 24 hour culture of Pseudomonas diminutia (100CFU/ml)  in a liquid medium to check the efficiency of the filtration system. Also, perform Bubble Point test before & after filtration.

Fill media aseptically performing all procedures identical to regular manufacturing process except pre & post purging of sterile nitrogen during filling

After filling, the filled units should be briefly inverted and swirled after filling to assure closure contact with the medium.

After Process simulation, thoroughly wash/ clean, disinfect & fumigate the area for two consecutive days.

4.4.5 Process Simulation of Sterile Dry Powder injectable

Set the powder filling weight range between 250-320mg, 500-625mg, or 1000-1350mg and fill the sterilized vials with sterile PEG 8000.

Check the weight of the filled PEG 8000 vials for 250mg, 500mg, and 1g vial after every 45 minutes as per SOP-04-025

Fill the powder-filled vials with 5.3ml sterile Tryptone Soya Broth by using a sterile syringe and then plug & seal the vials under a laminar airflow hood.

After filling, the filled units should be briefly inverted and swirled after filling to assure closure contact with the medium.

After Process Simulation, thoroughly wash/clean, disinfect & fumigate the area for two consecutive days.

4.5 Microbiological Monitoring

4.5.1 Perform monitoring of Air Changes & Air Velocity of Laminar Flow Hood (LFH) and Air Flow Pattern.

4.5.2 Perform sterility testing of filtered nitrogen and compressed air used in the filling,

4.5.3 Expose the settle plates at specified locations for 4 hours at rest (before filling) & at operational (during filling)

4.5.4 Perform swab test at rest (before filling) & at operational (during filling)

4.5.5 Perform non-viable particle count at rest (before filling) & at operational (during filling)

4.5.6 Perform viable particle count by using centrifugal air sampler at rest (before filling) & and at operational (during filling)

4.5.7 Check the microbial status of the finger of operators after filling. This activity should be performed outside the filling room.

4.5.8 Check the microbial status of the clothing of the operator after filling. This activity should be performed outside the filling room.

4.6 Incubation and Inspection

4.6.1 Transfer all filled vials to QC for incubation and inspection

4.6.2 Incubate the filled units at 30-35ºC for 7 days and then incubate at 20-25ºC for the next 7 days.

4.6.3 Periodically check the filled units for the growth of microbes in NLT 2000 Lux Light.

4.6.4 NMT 0.1% contamination should be observed

4.7 Failure investigation and Corrective action

4.7.1 A contaminated container should be examined carefully for any break in the container system

4.7.2 All positives (from integral containers) should be identified to at least genus, and to species whenever possible

4.7.3 The identification of contaminant should be compared to the database of the organisms identified in microbiological monitoring.

4.7.4 The biochemical profile of the contaminant can then be compared to that of microorganisms obtained from the sterility tests and bioburden and environmental monitoring programs, in order to help identify the potential sources of the contaminant.

4.7.5 Investigate sterility test procedures and room sanitation and sterilization methods to eliminate the cause.

4.7.6 Review monitoring technique for a possible problem.

4.7.7 Review monitoring technique for possible problem

4.7.8 Critical systems (HVAC, compressed air and gas, water, steam) should be investigated.

4.7.9 Calibration records should be checked

4.7.10 All HEPA filters should be investigated and rectified if warranted

4.7.11 Training records for all individuals (production, maintenance, cleaning) involved in the fill should be reviewed to assure proper training was provided

4.7.12 If the root cause is assignable, corrective action needs to be taken and documented.\

4.8 Documentation

4.8.1 The document should include at least the following:

  1. Identification of the process to be simulated and a copy of the batch record to be used.
  2. Identification of the class of the filling rooms to be used
  3. Identification of the filling line and equipment to be used
  4. Type of container and closure to be used
  5. Line speeds (low, normal, and high)
  6. Number of units to be filled
  7. Number and type of interventions to be included in the test
  8. Number of personnel participating
  9. Media or placebo materials to be used
  10. The volume of medium to be filled into the containers
  11. Incubator identification, and incubation time and temperature for the filled units
  12. Incubator identification, and incubation time and temperature for the filled units
  13. Environmental monitoring to be performed
  14. Growth promotion tests
  15. Box and tray number and time, especially of any positive units
  16. Verification of medium sterility

5.  Specifications

NMT 0.1% contamination on media-filled units should be observed.




7.1 FDA, Guidance for industry, Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice, September 2004.


7.2 Syed Imtiaz Hider, Validation Standard Operating Procedures A Step by Step Guide for Achieving Compliance in the Pharmaceutical, Medical Device, and Biotechnology Industries, 2nd edition, Taylor & Francis Group, LLC, USA 2006


7.3 Validation of Aseptic Processes (PI 007-51 July 2009), Section 4.3.1

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