INTERTECH

ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES

LABS
Protocol No:

Aseptic Media Fill

Validation Protocol
 INTERTECH
ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES
LABS
Protocol No:

TABLE OF CONTENTS
S.No Content Name Page No.
1. Protocol Approval

2. Objective
3. Scope
4. Abbreviation &Responsibilities
5. Definitions
6. Media Fill Training And Reference SOP/ STP and Equipment’s
7. Procedure
8. Methodology of Validation
9. Destruction Record
10. Reconciliation
11. Documentation
12. Conclusion
13. Re validation
14. Final Report
15. Reference
16. Revision History
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ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES
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1.0 PROTOCOL APPROVAL

Prepared By:

DEPARTMENT NAME SIGNATURE DATE

Microbiology

Reviewed By:

DEPARTMENT NAME SIGNATURE DATE

Production

Quality Control

Quality Assurance

Approval By:

DEPARTMENT NAME SIGNATURE DATE

Quality Assurance
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2.0 OBJECTIVE:
2.1. The objective of process simulation is to demonstrate the capability of Aseptic Process to
produce sterile drug products, certify aseptic processing equipment, process of filling
evaluate impact of planned and un-planned Interventions that take place during the normal
production and thus comply with current Good Manufacturing Practice requirements.
2.2. The objective of this protocol is to ensure that the aseptic filling process of the Ampoules on
the Ampoule Filling and Sealing Machine is under control and confirms to acceptability of
the aseptic filling procedure in protecting the product from adventitious microbial
contamination.
3.0 SCOPE:
3.1 This Protocol is applicable for validation of aseptic filling process of Ampoule filling line at
INTERTECH LABS
4.0 ABBREVIATION& RESPONSIBILITIES:
Abbreviation Definitions
BSCP Buffered Sodium Chloride Peptone
SCDM Soyabean Casein Digest Medium
FTGM Fluid Thioglycollate Medium
IPA Isopropyl alcohol
ATCC American Type culture collection
LAF Laminar Air Flow
BSC Biosafety Cabinet
-Ve Negative control
+ Ve Positive control
H Holiday
IP Indian Pharmacopoeia
USP United States Pharmacopeia
IPR Injectable Production
QA Quality Assurance
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Protocol No:
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ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES
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Protocol No:

4.1 The following personnel are responsible for the execution of the approved validation protocol.

Department Responsibility
 Preparation of the protocol.
 Carryout sampling and testing as per the protocol. Filled Ampoules inspection and
reporting.
Microbiology
 Detecting the organism in the Ampoule showing microbial growth (if any) OOS
investigation in case of failure Container closure integrity test.
 Destruction of media filled vials.
 Review of the protocol/report.
 To arrange for the prerequisites Ensure sampling as per protocol.
 Carrying out the aseptic filling operation as per Batch manufacturing record and
Production relevant SOPs.
 Cleaning and sanitization of the area and equipment’s before and after media fill
as per protocol and Participation in failure investigation.
 Destruction of the media filled Ampoules after approval from QA.
Ware House  To dispense the primary packaging materials and raw material as per the requirement.
 Review of the protocol / report.
 Ensuring availability of all related utilities.
Engineering  Carrying out engineering Interventions (if required) during media fill runs.
 Participating in the media fill runs.
 Participating in failure investigation.
 Review approval of protocol and report.
 Participate and monitor media fill.
Quality  Review of sterilization records & line clearance at all stages.
Assurance  In processes checks during media fill as per BMR and protocol
 Co-ordination with Quality Control for incubation and inspection.
 Failure investigation and corrective actions (if any).
Technical
 Protocol training for validation team.
Director
 INTERTECH
ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES
LABS
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5.0 DEFINITIONS:
5.1 Aseptic filling:
Part of aseptic processing where a pre-sterilized product/ media is filled and/ or packaged in
the sterile containers and closed
5.2 Aseptic processing:
Operation whereby the product/ media is sterilized separately then filled and packaged using
sterilized containers and closures in critical processing area.
5.3 Aseptic processing area (APA):
Controlled environment consisting of several areas in which, the air supply, materials,
equipment and personnel are regulated to control microbial and particulate contamination to
acceptable levels
5.4 Interventions:
An aseptic manipulation or activity that occurs at the critical area either it, should be
Planned/unplanned.
5.5 Routine Interventions:
Operator action that is known to occur during regular operations in the class 100 Ampoule
filling areas that are required to maintain the aseptic filling process. All known Routine
Interventions must be performed at every media fill
5.6 Non Routine Interventions:
Unexpected Interventions like jams, machine breakdowns etc.
5.7 Environmental Monitoring Program:
Defined documented Program, which describes the routine particulate and microbiological
monitoring of processing and manufacturing areas, and includes a corrective action plan
when action levels are exceeded.
5.8 Growth Promotion Test:
Test performed to demonstrate that media would support microbial growth.
5.9 Sampling Frequency:
Established period for collecting samples.
5.10 Sterilization:
A process, by which all viable microorganisms are removed or destroyed, based on Probability.
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5.11 Shift:
Scheduled periods of work or production, usuallylessthan8hours in length, staffed by
alternating groups of workers.
6.0 MEDIA FILL TRAINING AND REFERENCE SOP/ STP AND EQUIPMENTS:
6.1 Training:
The training of the testing plan and acceptance criteria for media fill of aseptic process to the
personnel responsible for execution shall be provided before execution and a training format
shall be filled in coordination QA department and Attached to Media fill validation report.

6.2 Validation Team:

S.No. Name Designation Responsibility Sign and date
1. Preparation of the protocol.
1.
2. Monitoring of Media fill Validation.
1. Inclusions of exclusions and related
2. actions of production.
2. Monitoring of Media fill Validation.
1. Review for correctness and
3. Completeness.
2. Monitoring of Media fill Validation
Validation team shall be responsible for the Training of media fill protocol provided as per
approved protocol.

6.3 Execution/ Working Team:

Execution or Working Team shall be responsible for the execution of media fill as per
approved protocol. The execution team is comprises of:
6.4 Training for Execution:
Quality Assurance team and other supporting personnel shall be trained for the execution of
aseptic simulation study as per media fill protocol
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6.4.1 Identification of Equipment’s and Rooms to be used:

S.No Room Name Room Number
1. Raw Material Dispensing Room 1.
2. Approved Primary Packing Material Storage 2.
3. Container Closure Preparation 3.
4. Compounding Room 4.
5. Filtration Collection Room 5.
6. De-cartoning Room 6.
7. Ampoule and Sealing Filling Room. 7.
8. Optical inspection Room 8.
List of Equipment’s
S.No Name of The Equipment Name Equipment No
1. Autoclave 1.
2. Ampoule Filling and Sealing machine 2.
3. Dry Heat sterilization DHS 3.
4. Garment cubicle 4.
5. Sterile Grade Filter 5.
6. 50.0 L Compounding vessel 6.
7. 50.0 L Filling vessel 7.
8. AHU 8.
9. Incubator (20 – 25°C) 9.
10. Incubator (30 – 35°C) 10.
11. MOBILE LAF (Cool zone) 11.
12. Dynamic Pass box (Compounding Room) 12.
13. Dynamic Pass box (Sterilization) 13.
14. LAF Compounding Room 14.
15. LAF (Vertical) – Ampoule Filling 15.
16. Dispensing Booth 16.
17. Weighing Balance (10 mg to 220 g) 17.
Water system :
S.No Name of The Equipment Name Equipment No
1. Water for injection 1.
2. Pure steam generations system 2.
Ampoule
S.No Material name Manufacturing /Supplier Sizes Quantity ( for each trail)
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ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES
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6.5 The following reference SOP/STP shall be used media fill Attach the copies of SOP/STP

S.No SOPs/ STPs Tittle SOPs/ STPs Number

1. Sterile area Entry & Exit procedure. 1.
2. Personnel entry in to factory. 2.
3. Preparation of 70 % iso propyl alcohol 3.
4. Selection and use of cleaning agents and disinfectants and their 4.
concentrations at sterile area and non sterile areas.
5. Transfer of the inspected vials and ampoules from quarantine 5.
area to packing area
6. Ampoule washing machine.

7. Ampoule filling & sealing machine

8. Unloading and storage of the sterilized materials in sterile area. IPR/051
9. Cleaning procedure for compounding room. IPR/042
10. Cleaning procedure for compounding room IPR/042
11. Moist heat sterilizer IPR/003
12. Dry heat sterilizer IPR/004
13. Assembling of the membrane filter assembly IPR/014
14. Leak test for ampoules and vials IPR/009
15. Visual inspection of filled vials/ampoules IPR/010
16. Media growth promotion, inhibition and sterility check MB/024
17. Sterility test method GTP/MB/001

7.0 PROCEDURE:
7.1 Pre- requisite Checks
7.1.1 Prior to taking up the validation of aseptic filling it should be ensured that all the following
area and equipment water systems and components have been qualified, validated and
approved.
7.1.2 Obtain the line clearance before starting filling operation of the batch for any leftover raw
material ampoules and etc of previous day/batch.
7.1.3 Check the area and machine for cleanliness.
7.1.4 Check the temperature, differential pressure and relative humidity of vial filling room.
7.1.5 Take containers of sterile SCDM as per requirement of batch manufacturing record.
7.1.6 Wipe the sterile SCDM container with 70% IPA. Transfer the container in dynamic pass box
under UV light contact for 15 minutes before taking the SCDM container in aseptic area.
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7.1.7 Transfer the sterile SCDM into blender and mix them for 30 minutes for each batch, after that
transfer the blended sterile SCDM powder in pre sterilized container used for filling activity.
7.1.8 The above-mentioned activity is performed to simulate the actual condition during routine
Production.
8.0 Methodology of Validation

8.1 Process Flow

8.2 Preparation and Qualification of media
8.3 Sterilization of Accessories
8.4 Sterility of the Components
8.5 Process Simulation
8.6 Interventions during Media Fill Run
8.7 Monitoring During the media fill Validation
8.8 Inspection of the Filled Ampoules
8.9 Incubation Control
8.10 Post Incubation Growth Promotion Test
8.1 PROCESS FLOW
8.1.1. Process Flow& Procedure:
8.1.1.1. Flow procedure below

Media Fill

Media
Preparation
(SCDM)

Media Transfer from

Microbiology Section to
Ampoule Filling Area with
Precautions

Machine Ready for Media

filling

Line Clearance from QA for

Media filling
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ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES
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Media fill shall be by the

filling Operator

Ampoule Sealing

Visual Inspection

Incubation

20-25 C 30 -25 C 42 - 44 C
(Fungal) 14 (Bacteria) 14 (Fecal
Days Days Organisum) 14

8.1.2. Media fill record:

8.1.2.1. Process simulation will be performed as per Batch manufacturing record. BMR
No.__________
8.1.3. Identification of Rooms to be used:
8.13.1. Batch manufacturing is done in the Compounding Room and media is filtered through
0.22,m filter under LAF in the aseptic processing area and the media is filled into Ampoule
Under LAF in the Ampoule filling room.
8.1.4. Ampoule filling sealing operation:

8.4.1.1. Ensure that the nitrogen gas valve are open and connected to the Ampoule filling and
sealing machine. The pressure is within the acceptable range. Switch on the Machine. All the
sterilized accessories like needle with silicon tubing’s is connected to their respective positions.
Filtered media in a sterile stainless steel pressure vessel shall be brought under Laminar Air
Flow Unit and connected to the peristaltic pump. All the activities shall be carried out
aseptically.
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8.4.1.2. Ensure that the washing and sterilization activity has been started at least two hours
before starting ampoule filling activity. Wait until the sterilized Ampoules are reached to the
table of ampoule filling machine.
8.1.4.3. Aseptically connect the silicone tubing’s 4 individual syringes fitted in the syringe rack.
8.1.2.4. Fix the nozzles to the filing turret.
8.1.2.5. Aseptically connect the outlet of the syringes to the filling nozzles with silicone tubing’s.
8.1.2.6. Connect the outlet silicone tubing of the SCDM containing Vessel.
8.1.2.7. Allow the SCDM to the syringes and to the nozzles.
8.1.2.8. Carryout the filling activity as per Aseptic Media fill SOP& BMR.
8.1.2.9. Collect the SCDM sample from SS vessel container as per sampling plan and send to QC
along with In-process sample Request.
8.1.2.10. Adjust the required volume individually and check the volume in the Ampoule.
8.1.2.11. Carry out the filling and sealing activity and simulate the Interventions and record the
details.
8.1.2.12. Check the media filled and sealed Ampoule for proper Sealing.
8.1.2.13. Collect the filled and sealed samples as per sampling plan and send to QC along with
In-process sample request.
8.1.2.14. Collect the left over media sample at the end of filling of each container as per sam-
pling
Plan along with In-process sample request.
8.1.2.15. Check the sealed Ampoule for proper sealing.
8.1.2.16. Label the trays with required details.
8.1.2.17. Collect the sealed Ampoule in the labeled trays.
8.1.2.18. Collect the samples of sealed Ampoule as per sampling plan and send to QC along with
In-process sample request. Record the details in the Record of Results.
8.1.2.19. During the media fill perform the environmental monitoring record as per annexure-III
8.1.2.20.. Enter the all Interventions routine and non routine in annexure-II.
8.1.2.21. Start the filling and sealing activity. Check for the volume and sealing quality intermittently
and collect ampoules for incubation at the interval of 15 minutes from each filling head
( Rest of the ampoules are to be stored in the temperature 20-25°C for 7 days and further at
30-35°C for 7 days in a controlled room).Collect the filled and sealed Ampoules in tray
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ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES
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and pass the filled tray through pass box to the ampoule collection area (Ampoules
Inspection area)

8.1.5. In the Filtration and material movement area:

8.1.5.1. Two persons from Production for the addition of assembly of filling equipment is the normal
requirement. Additionally, a microbiologist will be there for microbiological Monitoring.
8.1.5.2. During the media fill, Interventions situation one person from Engineering, one person
from or from production is added as extra one person.
8.1.6. In the Ampoule sealing area:
8.1.6.1. One persons from Production for the addition of assembly of sealing equipment is the
Normal requirement. Additionally, a microbiologist will be there for microbiological
monitoring.
8.1.6..2. During the media fill, as Interventions situation one person from Engineering, one
person
From QA or from production is added as extra one person.
8.2. PREPARATION AND QUALIFICATION OF MEDIA:
8.2.1 Selection of the Media:
8.2.1.1. Soyabean Casein Digest Media (SCDM) shall be used for media fill.
8.2.1.2. Dehydrated Soyabean Casein Digest medium (SCDM) of TM MEDIA will be used.
Justification for using sterile SCDM to simulate the filling process:
1. Low selectivity, clarity, media concentration and filterability.
2. Solubility of SCDM in water.
3. Flow ability of SCDM through the filling needle.
4. Ability to support growth of a wide range of microorganisms.
5. It does not inhibit the growth of microorganisms at the selected concentration.
8.2.2. Preparation of Media:
8.2.2.1.Prepare and sterilize SCDM media as per batch manufacturing record readymade media
after checking the appearance and the expiry date shall be used5 litter media prepared.
8.2.2.2. The percentage of the media shall be 3 %.
8.2.2.3. Weigh and dissolve the medium in about half of the required quantity of Purified water
and after dissolving make up the volume with same water.
6.2.2.4. The media completely dissolve and clear of the media .
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8.2.2.5.5 litter media prepared Check and record the pH. SCDM medium with pH of 7.3 ± 0.2
shall be used.
8.2.2.6. Make adjustment in the pH with 0.1N NaOH or 0.1N HCl solutions if required.
8.2.2.7. Use clean dried glass containers for media preparation.
8.2.2.8. Record the observations in the ‘Media Preparation Record’. The format for Media
Preparation Record is attached as Attachment .
8.2.3. Preparation and Sterilization of SCDM:
8.2.3.1. Media sterilization as per manufacturing recommendation and as per sop no MB/003.
8.4.4. Dispensing of SCDM:
8.4.1.1. Dispense the material (Media) as per media fill batch manufacturing record.
8.4.1.2. Dispense required Quantity of SCDM powder in SS container and transfer into production
area.
8.4.1.3. Dispense required quantities of 2 mL glass Ampoule, in suitable containers, and
transfer into production area.
8.2.5. Filtration of the Media:
8.2.5.1. The media solution shall be filtered through a pre sterilized 0.22µ membrane filter into a
previously sterilized SS pressure vessel under Laminar Air Flow Unit.
8.2.5.2. The integrity of the sterilized filter should be confirmed immediately after use as per
SOP No IPR/014The result of the integrity test shall be entered in the record
8.2.6. Growth Promotion Test of The Filtered Media:
8.2.6.1. 100 ml of the sterilized media shall be collected in sterile container (3 no) for Growth
Promotion Test. The Growth Promotion Test shall be carried out with As per gillus niger
(ATCC 1344 ), Bacillus subtilis (MTCC 441 ) and Candida albicans (MTCC 227 ).
8.2.6.2. The Growth Promotion Test should demonstrate that the medium supports recovery and
Low number of microorganisms ie. 10-100 cfu / unit or less.
8.2.6.3. Prepare culture suspensions as per SOP No.MB/023 .and select suitable dilution and
volume that gives 10-100 cfu / unit or less.
8.2.6.4. Observe the medium daily for growth.
8.2.6.5. The test medium is valid, if it shows adequate growth within specified incubation period.
8.2.6.6. The medium is considered invalid if it shows inadequate growth response.
8.2.6.7. Record the observations in the ‘Growth Promotion Test Record’. The format for Growth
Promotion Test Record is attached as Annexure.
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8.3. STERILIZATION OF ACCESSORIES:

8.3.1. Sterilization of Filling & Sealing machine parts (Filling needle, Silicone tube), SS
forceps, etc shall be done before commencing Media filling.
8.3.2. The sterilization of accessories shall be carried out as per SOP No. IPR/03.
8.4. STERILITY OF COMPOUNDS:
8.4.1 Ampoules shall be collected separately in sterile containers just before the start of the
filling operations to confirm the sterility of the components.
8.4.2 The test shall be carried out as per SOP No. GTP/MB/001.Result of the test shall be
entered in the Data record.
8.5. PROCESS SIMULATION.
8.5.1. Type of container and Closures to be used:
8.5.1.1. 2 ml Clear Ampoule.
8.5.2. Line Speed:
Ampoule Size Line Speed Qualified Line Speed for media fill
2 ml clear Ampoules 60 – 120 Ampoule Per Minute Ampoule Per Minute -70 No’s
8.5.3. Equipment’s to be used for simulation:
8.5.3.1. Compounding/Mixing vessels, Filling/Holding vessels and entire filtration and assembly
will be the same which will be used for regular product manufacturing.
8.5.4. Line Setup:
8.5.4.1. The filling line setup includes of filtration assembly, filling assembly, sealing assembly.
8.5.5. Duration of Fill:
8.5.5.1. Media fill will be done in General shifts (Approximate-08 hrs) with intermittent
Stoppages for covering all Interventions.
8.5.6. Number of units to be Filled (For Incubation) and Volume of Medium to be filled:
8.5.6.1. Media Fill 5500 Ampoule with sufficient quantity of filled medium5 Littre medium
prepared.
Min. volume of medium to be
Size of the Ampoule
filled

8.5.7. Processing of media fill:

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ASEPTIC MEDIA FILL VALIDATION PROTOCOL FOR AMPOULES
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8.5.7.1. The filling and sealing machine shall be operated as per the SOP No IPR/007
8.5.7.2. Filtered media in the SS vessel shall be brought under the Laminar Air Flow Unit of the
filling and sealing machine.
8.5.7.3. The fill volume of the vial to be set for targeted fill volume to deliver 1 ml (for 2 ml amp.)
8.5.7.4. The filling operation is started and the Ampoules are filled with media solution.
8.5.7.5. The filled Ampoules are then sealed on line.
8.5.7.6. Check the quality of sealing on line.
8.5.7.7. The filled and sealed Ampoules are transferred through pass box outside the aseptic filling
area.
8.5.7.8. Collect the Ampoules in SS trays.
8.5.7.9. Each tray shall be labeled with Run Number / Session / Serial Number / Number of
Ampoules Filled Date and Sign.
8.5.8. Filling components and other materials preparation & sterilization:
8.5.8.1. Prepare and sterilize the filling components and other materials as per SOP IPR/053
8.5.8.2. Perform activity and record the activity as per batch manufacturing record.
8.5.9. Washing of Ampoule:
8.5.9.1. Wash the Ampoule as per SOP by using Ampoule washing machine.
8.5.9.2. Sample the Ampoule randomly and check for clarity of filtered water rinsed from
washed Ampoule for as per SOP IPR/007 and record the details batch manufacturing
record.
8.5.9.3. Collect the samples of empty washed Ampoule as per sampling plan.
8.5.9.4. Collect the samples of empty depyrogenated Ampoule as per sampling plan.
8.5.10. Transfer of sterilized filling components, Ampoule and SCDM:
8.5.10.1. Transfer the sterilized filling components into Ampoule filling area through Mobile LAF.
8.5.10.2. Transfer the sterilized Ampoule in to Ampule filling Machine.
8.5.10.3. Transfer the Filtered SCDM Vessels from filtration room to filling area.
8.5.11. Number of Media Fill Runs & Filling Quantity:
8.5.11.1..Media fill shall be conducted in General shift in 08 hrs.
8.5.11.2. Media fill shall be carried out with Soyabean Casein Digest Medium.
8.5.11.3. Size of media fill 5000 ampoules
8.5.11.4. Media fill shall be covering two shifts and considering all possible interventions.
8.5.11.5. Filling speed and Filling Session their records shall be maintained during media fill.
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Table 1.
Table 1
S.No Interventions Ampoule Time/ session Number of Total Tray /
size Ampoules To Be Filled No

Remarks:
Done by: Checked by:
8.5.12. Discard Policy:
8.5.12.1. Destroy the rejected media filled Ampoule as per IPR/031.
8.5.12.2. Destroy the media filled Ampoule after completion of incubation period as perSOP
IPR/031.
8.5.13. Media remaining in the filling tank:
8.5.13.1. Left over material shall be destroyed as per SOP IPR/031.
8.5.14. Number of Personnel Participating In the Filling area:
8.5.14.1. Persons (Supervisors and Operators) from different departments like Production,
Quality
Assurance, Quality Control, Maintenance shall be present – by rotation in trials to cover all the
persons. Minimum number of persons permitted at a time during operations shall not be less 6
8.5.14.2. Two persons from Production for the addition assembly of filling equipment is the
normal
requirement. Additionally, a microbiologist will be there for microbiological monitoring.
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8.5.14.3. During the media fill, Interventions situation one person from Engineering, one person
From QA or from production is added as extra one person.
8.5.15. Garments:
8.5.15.1. All the gowns used during the media fill runs shall be sterilized at least one day prior to
the media fill .
8.6. INTERVENTIONS DURING MEDIA FILL:
8.6.1. Planned Interventions and Un Planned Interventions during following interventions shall be
Covered in the entire media fill:
8.6.1.1. Failure of Power Supply for two minute.
8.6.1.2.Change of Operator.
8.6.1.3.Break down of the filling machine.
8.6.1.4.Removing Ampoules for fill volume check.
8.6.1.5.Changing of hand gloves.
8.6.1.6.Others
8.6.1.7.Record all the observations in the ‘Planned Interventions and Un Planned Interventions
Record’. The format for ‘Planned Interventions and Un-Planned Interventions Record’ is
attached as Annexure No.
8.6.2 .Planned Interventions(Routine):
8.6.2.1. The following normal activities, which occur during routine aseptic filling process,
will be included in the process simulation.
8.6.2.2. All the Interventions are categorized as routine and non-routine Interventions. Routine
Interventions are those which occur commonly and Non routine Interventions are those
which occur rarely.
8.6.2.3. All the routine Interventions will be simulated in each media fill. While non routine
Interventions will be simulated at least annually.
S. No. Interventions(Routine) Rational
Removal of rejected Ampoule from This is normal activity which occurs during
1.
collecting tray. normal production.
Adjustment to filling pumps/nozzles - This is normal activity which occurs during
2.
Routine normal production.
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S. No. Interventions(Routine) Rational

Removing Ampoule fill volume check.
This is normal activity which occurs during
3. (Only Simulate not to remove actual
normal production.
Ampoule)

Adjust the filling speed of machine at This is normal activity which occurs during
4.
minimum/Maximum. normal production.

This is normal activity which occurs during

5. Ampoule Filling Machine breakdown
normal production

Simulating changeover of power from SEB This is normal activity which occurs during
6.
to generator for two minutes normal production

This is normal activity which occurs during

7. Change of Operator
normal production

S.No. Interventions(Non Routine) Rational

To demonstrate the ability of operator to correct

1. Replacement of filling pumps/nozzles.
the problem without contamination.

To demonstrate the ability of engineer to correct

2. Replacement of Sensors By Engineer.
the problem without contamination.

Removal of Sample from filling container To demonstrate that the sampling doesn’t have an
3.
using sampling valve impact on sterility of solution.

8.6.3. Un-planned Interventions:

8.6.3.1. The all Un-Planned Intervention shall be captured in Batch Manufacturing record.
8.6.3.2 Sampling Plan Interventions(Routine)
S.No. Interventions(Routine) Sampling Quantity
1 Removal of rejected Ampoule from collecting tray. 5 No's
2 Adjustment to filling pumps/nozzles - Routine 5 No's
3 Removing Ampoule fill volume check. (Only Simulate 5 No's
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not to remove actual Ampoule)

4 Adjust the filling speed of machine at 5 No's

minimum/Maximum.

5 Ampoule Filling Machine breakdown 5 No's

6 Simulating changeover of power from SEB to 5 No's

generator for two minutes

7 Change of Operator 5 No's

8.6.3.3 Sampling Plan Interventions(Non-Routine)

S.No. Interventions(Routine) Sampling Quantity
1 Replacement of filling pumps/nozzles. 5 No's

2 Replacement of Sensors By Engineer. 5 No's

3 Removal of Sample from filling container using 5 No's

sampling valve

8.6.3.3 The Sampling to be labeled and collected in Polythene bags for each intervention and
transfer with precautions. The Media fill ampoules to be incubated in labeled Petri dish/ Petri
Plates as per Process flow.
8.7. MONITORING DURING THE MEDIA FILL VALIDATION
8.7.1 Environmental Conditions Monitoring:
8.7.1.1. Monitor the environmental conditions for temperature, differential pressure and relative
humidity.
8.7.1.2. Record the observations in the ‘Environmental Conditions Record’. The format for
‘Environmental Conditions Record’ is attached as Attachment 2
8.7.2. Personnel Monitoring:
8.7.2.1. Ensure that personnel entering the area have followed the gowning and entry procedures as
per SOP No. IPR/015
8.7.2.2. Personnel monitoring shall be carried out as per SOP No IPR/065.
8.7.2.3. During personnel monitoring if any organisms are observed, the same organisms shall be
isolated and preserved in the Quality Control (Microbiology) Laboratory for Identification.
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8.7.3. Viable particle monitoring:

8.7.3.1. Microbial monitoring should be performed in and around areas of high operator activity.
8.7.4. Plate exposures:
8.7.4.1. Plate exposures shall be done during media fills as per sop IPR/065
8.7.5.1.Surface swabbing shall be done at the end of filling activity as per sop IPR/066
8.7.6. Personnel swabs:
8.7.6.1. Personnel should be monitored as below through swabs as per sop.IPR/065
8.7.6.2. Every time when the Personnel leave Aseptic core area,
8.7.6.3. Whenever they are involved in attending the critical Non routine Interventions filling and
Sealing operations.
8.7.6.4. Additionally, Microbial monitoring will also be done in adjacent aseptic areas while
media fill is going on even there is no activity.
8.8. INSPECTION OF THE FILLED AMPOULES.
8.8.1. Inspection:
8.8.1.1. Inspection of the filled and sealed Ampoules shall be done by qualified personnel as per
SOP No. —
8.8.1.2.Rejection analysis shall be done and reason of rejection to be identified and recorded.
8.8.1.3. Record all the observations in the ‘Inspection of Filled Ampoules Record’. The format
for ‘Inspection of Filled Ampoules Record’ is attached as media fill BMR.
8.8.1.4.100 % inspection to be carried out prior to incubation.
8.8.1.5.Media filled Ampoule will be manipulated by inverting to allow contact of the medium
with all product contact surfaces i.e. inner surface of the container and closure before
Inspection.
8.8.1.6.Media filled Ampoule will be inspected after filling & sealing, 7 days and 14 days of
Incubation.
8.8.1.7.Leaking or damaged media filled Ampoule will be removed and a record made of such
removal, following filling and prior to incubation of the media.
8.8.1.8. Each media filled unit should be examined for contamination by personnel with
appropriate
education / training and experience in inspecting media fill units for microbiological
contamination. If this activity is performed by trained microbiologist or trained operator
then it should be supervised by QA.
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8.8.1.9. To comply with the requirement that the volume of media filled must be sufficient to
wet all surfaces including the closure and to allow for easy inspection, as control limits at filling
are set up very widely to allow doses rejects, some Ampoule may contain some drops of
broth.
8.8.1.10. For these low filled items, the level has been defined with microbiological laboratory to
Enable final inspection of Ampoule the minimum level of media inside the Ampoule must
entirely cover the glass bead when the Ampoule is standing up. This minimum level
allows inspection of the Ampoule after incubation and detection of microbiological growth.
8.8.1.11. All suspect units identified during the examination should be brought to the immediate
attention of the In-charge microbiologist.
8.8.1.12. An identification of the organism will be performed, but the information will most
likely be of little value for damaged containers.
8.8.2. Incubation and Incubation Temperature and Time for the Filled units:
8.8.2.1. Incubate all the filled Ampoules except the ones having sealing defects, develops cracks
during the filling and rejected during optical inspections.
8.8.2.2. Invert and swirl to ensure that all the internal surface are in contact with media.
8.8.2.3. Incubate the filled good Ampoules for 7 days at 20-25°C.
8.8.2.4. After 7 days, inspect the Ampoules for contamination. If all the Ampoules are clear invert
and swirl to ensure that all the internal surface are in contact with media.
8.8.2.5. The containers will be inspected for any microbial growth and then transferred to another
set of Incubate the Ampoules at 30-35°C for further 7 days.
8.8.2.6. Visually inspect the Ampoules for growth of microorganism against black and white
back ground and record in validation report.
8.9. POST INCUBATION GROWTH PROMOTION TEST
8.9.1. Growth Promotion Test:
8.9.1.1. Growth Promotion test will be performed on media filled units after 14 days incubation
and inspection for the absence of microbial growth or contamination as per SOP:MB/024
The growth promotion units must be inoculated with <100 CFU challenge. Following
organisms will be used to demonstrate that the medium supports recovery and growth of
low numbers of microorganisms.
8.9.1.2. Bacillus subtilis MTCC#736 Equivalent to ATCC # 6633
8.9.1.3. Candida albicans MTCC#227 Equivalent to ATCC# 10231
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8.9.1.4. Staphylococcus aureus MTCC#737 Equivalent to ATCC# 6538

8.9.1.5. Escherichia coli MTCC#1687 Equivalent to ATCC# 8739
8.9.1.6. Pseudomonas aeruginosa MTCC#1688 Equivalent to ATCC# 9027
8.9.1.7. Aspergillus niger MTCC#1344 Equivalent to
ATCC#16404
8.9.1.8. Recent Environmental isolates preferably the organisms isolated during the media
filling. Growth promotion test units should demonstrate growth of the test organisms
within 3days for bacteria and 5 days for fungi, yeasts and molds for the media fill test to
be acceptable. The incubation temperatures will be 30 – 35ºC and 20 – 25ºC.
8.9.1.9. If the growth promotion testing fails, the origin of any contamination found during
the simulation should nonetheless be investigated and the media fill promptly repeated.
Use the environmental isolate obtained during the day of the media fill from the same
room for Growth Promotion Test (GPT). If isolates are not found in the same room
isolates from adjacent room or isolates obtained from previous days may be used for
GPT.
8.10. INTERPRETATION OF THE RESULT
8.10.1. After the incubation period of the media filled containers they are visually examined for
microbial growth. Contaminated containers should be examined for evidence of container
closure damage, which might compromise integrity of the packing system.
8.10.2. Observe the growth/turbidity in media filled vials, after the 7 and 14 days of incubation;
first incubate media filled vials at 20ºC to 25ºC for 7 days. After completion 7 days incubation
period visual checking shall be done for contamination of filled vials.
8.10.3. Another 7 days Incubation shall be carried out at 30ºC to 35ºC, at the end of incubation
period; visual checking shall be done for the contamination of filled vials. Note down the
temperature throughout incubation period and record.
8.10.4. Any Contaminated unit should be considered objectionable and investigated. The
microorganisms should be identified to species level. The investigation should survey the
possible causes of contamination. In addition, any failure investigation should assess the impact
on commercial drugs produced on the line since the last media fill.
8.10.5. Whenever contamination exists in a media fill run, it should be considered indicative of a
potential sterility assurance problem, regardless of run size. The number of contaminated units,
should not be expected to increase in a directly proportional manner with the number of units in
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the media fill run. Test results should reliably and reproducibly show that the units produced by
an aseptic processing operation are sterile.
8.11. ACCEPTANCE CRITERIA:
8.11.1. The target shall be zero contamination however the following criteria shall be applicable.
8.11.2. Microbial growth should not appear after 14 days of incubation.
8.11.3. Aim of the process simulation shall be zero contaminated units. When filling<5000units,
Then NO contaminated units should be detected.
8.11.4. One (1) contaminated unit should result investigation.
8.11. 5.Two (2) contaminated units are considered cause for revalidation following investigation.

Units of Filled Limit

< 5000 NIL

5000 to10,000 1

>10,000 1

8.13. FAILURE INVESTIGATION:

8.13.1. In case of failure of media fill validation following investigation shall be carried out.
8.13.1.1. For any run size, intermittent incidents of microbial contamination may be
indicative of low-level contamination that shall be investigated. Investigation of gross
failures should include the potential impact on the sterility assurance of batches manufactured
since the last successful media fill.
8.13.1.1. The interval between the washing and drying and the sterilization of components,
containers and equipment as well as between their sterilization and use should be minimized
and subject to a time-limit appropriate to the storage conditions.
8.13.1.1. Handling Components, containers and equipment after the final cleaning process in
such a way that they are not re contaminated.
8.13.1.1. Environmental monitoring shall be investigated which was carried out during media
fill Validation.
8.1.1.1. If system simulation run fails failure investigation shall be carried out as per
respective SOP

8.13.2. Microbiological identification:

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8.13.1.1. Identification of contaminated organisms. Compare identity of media fill

contaminating organism(s) to that of organisms isolated from as per sop MB/011
8.13.1.2. Review Microbial environmental monitoring data and confirm that appropriate
standards and complied with specifications.
8.14. Corrective Actions:
8.14.1 The media fill runs shall be repeated again until all the three consecutive runs comply.
8.14.2. The recording for corrective actions shall be carried out in the ‘Corrective Actions Report’.
The format for ‘Corrective Actions Report’ is attached as Attachment.
9.0. DESTRUCTION RECORD:
9.1. Media filled Ampoule without any contamination after final inspection will be destroyed as
per SOP/MB/006Ampoule used for growth promotion test and container closure test if any will
be destroyed after autoclaving at 121ºC for 30 minutes. If any Ampoule showing growth will be
destroyed by autoclaving after isolating the organisms from the Ampoule and document the
information Records for destruction will be maintained.
10. RECONCILIATION:
10.1.100% reconciliation/accountability of all filled units will be the target. Any excursion will
be investigated and documented; however, a variance will not be an automatic invalidation of the
media fill validation since it is impractical to expect perfect reconciliation of Ampoule using
high speed equipment.
11.0. DOCUMENTATION
11.1. This validation protocol will be executed through the following documents:
S. No. Attachment title Attachment/ Annexure number
1. Process Flow Chart.
2. List of Interventions. 9.
3. Environmental monitoring Record. 10.
4. Media preparation details 11.
5. Growth promotion test 12.
6. Sterility test 13.
7. Incubation details 14.
8. SOP/STP 15.
9. Media fill validation BMR 16.
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12.0. CONCLUSION:
12.1. Aseptic process simulation program is designed to simulate the production process and
incorporate defined “most challenging” conditions as appropriate and to validate the aseptic
condition of filling operations initially and for revalidation.
12.2. "Most challenging case" conditions were defined according to our knowledge of the processes
on this line, and according to an evaluation of manufacturing practices that might influence
the likelihood of microbial promotion.
12.3.This will validate the aseptic filling of the different products manufactured on Ampoule line.
12.4.Routine revalidation will occur at a frequency that is no less than twice per year at the most
challenging process.
13.0. REVALIDATION:
13.1 Revalidation shall be performed after any significant modification to the equipment area,
process, number of shifts and change in operators.
13.1. Frequency
13.1.1 Once in a year.
14.0. FINAL REPORT
14.1. Approval of the Validation Report will be the joint responsibility of the following:
14.2. The summary report shall prepared by Validation, and reviewed by Production, Quality
Control and Engineering. The Quality Assurance shall do the final approval of the report.
15.0. REFERENCES:
15.2. USFDA Guidelines for Sterile Drug Products Produced by Aseptic Processing Current
Good Manufacturing Practices.
16.0. REVISION HISTORY: