MEDIA FILL VALIDATION PROTOCOL

ASEPTIC MEDIA FILL

APPROVAL SIGNATURES

Signing of this Approval page indicate the agreement with the validation approach described in this document. Should modification be required an addendum shall be prepared, checked and approved.

Name/designation

Signature

Date

Prepared by

Microbiology

Checked by

Manager, Production

Asst., Manager, QA

Approved by

DGM – QA

CONTENTS

Sr No Index

A. Overview

1 Objective

2 Process Description of Vial Filling Operation

3 Brief Plan of Media Fill

4 Equipment Description

5 Container closure Systems

6 Revalidation Frequency

7 Responsibility

B.

Validation Methodology

1 Pre-validation Checks

2 Environmental Checks during Validation runs

3 Validation with Media

3.1 Sterilization of Lactose powder

3.2 Transfer of sterilized lactose container to critical area

3.3 Suitability test of lactose

3.4 Preparation of Media

3.5 Method of Growth Promotion Test

3.6 Component Preparation

3.7 Media Fill Operation

3.8 Growth Promotion Test of Lactose Filled Vials

3.9 Worst Case Consideration

3.10 Personnel Involvement

3.11 Incubation of Vials and observation

3.12 Destruction of Vials after incubation and observation

C Acceptance Criteria

D Action taken in case of System Simulation Test Failure

E Documentation

F Conclusion

INTRODUCTION

The sterile powder vial filling process involves the following sub processes.

· Washing and sterilization of rubber stopper direct sterilization of RFS rubber stoppers

· Steam sterilization of filling machine parts which directly comes in to contact with sterile powder (a part from vial & stoppers)

· Sterilization of garments, water filtration accessories, etc.

· Storage of material intactly under LAF.

· Transfer and handling of material in the aseptic area.

· Continuous closure integrity.

· Human handling and intervention during product processing.

· Being a sterile product and each validated sub processes is having its own limitations. Further the material handling and interventions during processing cannot be validated directly. Similarly the product produced is only sampled for microbial testing.

To ensure 100% testing at least on a limited quantity of vials aseptic media fill in considered to be the best way to evaluate the freeness of the product from microbial contamination.

A. OVERVIEW

1.0 OBJECTIVE

This protocol is designed to establish sufficient data to assure that the aseptic powder filling process is adequate and drug product thus produced remains sterile.

This protocol provides a standard procedure for the validation of aseptic dry powder filling process environmental condition and practices to confirm its acceptability in protecting the product from microbial contamination.

2.0 Responsibilities:

2.1 The pharmaceutical manufacturing group will be responsible for the following:

2.1.1 Conducting the outlined procedure.

2.1.2 Ensuring that requirements outlined in this procedure are met prior to ending a media fill run.

2.1.3 All events are completely and clearly documented on the appropriate attachments of this SOP.

2.2 The validations department will be responsible for the following:

2.2.1 Reviewing requirements for all media fills. The requirements will be documented in attachment 1.2. And 3.

2.2.2 Coordinating each media fill with the manufacturing group.

2.2.3 Submitting samples for growth promotion to microbiology.

2.2.4 Ensuring that the acceptance criteria per this procedure have been met, and preparing a final report.

2.3 The packaging department is responsible; for the visual inspection of the media fill vials.

2.3.1 The quality assurance department is responsible for approving requirements for all media fill prior to fill. The requirements will be documented in attachment 2.

2.4 The microbiology Department is responsible for:

2.4.1 Conducting environmental monitoring (EM) per CP-QC_MICRO-004.

2.4.2 Immediately reviewing the nonviable particle counts and notifying management if alert and/ or action levels are exceeded

3.0 PROCESS DESCRIPTION FOR VIAL FILLING OPERATION:

3.1 Vial Washing

3.1.1 Approved vials are decartoned in the decartoning room. The vials are placed into stainless steel boxes and transferred into vial washing and sterilization room.

3.1.2 The vials are loaded onto the washing machine conveyor. Washing of vials takes place in washing machine and the washed vials are delivered into tunnel sterilizer with the aid of conveyor.

3.1.3 The washed vials enter into the tunnel sterilizer under laminar airflow protection.

3.1.4 The washed vials enter the tunnel sterilizer for sterilization & Depyrogenation.

3.2 Vial sterilization & Depyrogenation

3.2.1 The tunnel sterilizer is a continuous belt type dry heat sterilizer having drying, sterilization, cooling and stabilization zones to facilitate sterilization and depyrogenation.

3.2.2 The washed vials first enter into the drying zone of the tunnel sterilizer where the residual water in the washed vial gets evaporated.

3.2.3 Then the vials enters into the sterilization zone where the vials gets sterilized and depyrogenated by hot HEPA filtered air (Class 100).

3.2.4 Then the vials enter into cooling zone and get cooled by HEPA filtered air (Class 100).

3.2.5 Finally the vial enters into the stabilization zone where the vials are further cooled by HEPA filtered air (Class 100) and finally delivered onto the vial filling machine turntable at a temperature of 23 to 250C under unidirectional Air Flow zone.

3.3 Rubber stopper sterilization and transfer to filling room

3.3.1 Washed rubber stoppers sterilization.

3.3.1.1 The approved rubber stoppers are washed in rotary plug washer and siliconised.

3.3.1.2 These rubber stoppers are loaded into the perforated cassettes of the bung processor under LAF (class 100). Each cassette is placed in the rotating carriage and the rotating carriage is loaded into the bung processor and subjected to steam sterilization and drying.

3.3.1.3 The rubber stoppers after sterilization and drying & cooling are unloaded into previously sterilized non-perforated rubber stopper holding canisters under mobile laminar air flow work –station (class 100)

3.3.1.4 These canisters are transferred into the sterile quarantine area of the respective sterile powder filling area and are stored under laminar airflow workstation.

3.3.1.5 The rubber stoppers stored in the canisters are transferred into the filling room via hatch, kept under LAF and are used at the time of filling.

3.3.2 Ready for sterilization rubber stoppers.

3.3.2.1 The rubber stoppers packed in Ready for sterilization packs shall also be simulated.

3.4 Raw material transfer to filling area

3.4.1 Approved sterile RM is transferred from stores.

3.4.2 Kept the hatch & disinfected.

3.4.3 Transfer canisters to API transfer room with LAF & dynamic pass box.

3.4.4 Disinfect containers, keep in dynamic pass box.

3.4.5 Unload into aseptic area.

3.4.6 Store under LAF.

3.4.7 Transfer using mobile LAF to dynamic pass box opening into filling area.

3.4.8 Unload into filling room through dynamic pass box and transfer to filling machine.

3.5 Filling and stoppering

3.5.1 The filling machine is located in the class 100 zone. The machine is provided with enclosure providing class 100 A environment on the filling area and 100 for operating the machine.

3.5.2 The filling machine will be fitted with suitable change parts like indexing wheels with suitable pistons, powder hoppers and rubber stopper hopper after steam sterilization and drying.

3.5.3 Filling machine is operated as per the standard operating procedure.

3.5.4 In process sampling during filling operation is carried out as per the standard operating procedure

3.6 Sealing

3.6.1 The filled and stoppered vials thus transferred from the filling room are allowed onto the sealing machine track.

3.6.2 The sealing of the vials carried out as per the standard operating procedure.

3.7 Filling area

3.7.1 The filling room is class 100 area. The filling & stoppering machine is protected by an enclosure with doors suitable for machine operation. The filling zone is class 100A and the other surrounding area out side the filling zone is class 100B.

3.7.2 The filling area is constructed with SS panels. The flooring is painted with self-leveling epoxy painted.

3.7.3 Nitrogen/carbon dioxide and vacuum required for filling are supplied into sterile area from a pendent through sterilizing grade filters. A provision of compressed air instead of nitrogen is provided for media fill operation

3.8 Environmental controls

3.8.1 Temperature of filling area is maintained not more than 250C

3.8.2 Relative Humidity of filling area is maintained not more then 30% ± 5%.

3.8.3 Pressure differential of the filling room is maintained at 0.5 mm of water column higher when compared to sterile corridor and a minimum of 1.5 mm of water column higher compared with vial sealing and vial washing sterilization zone.

3.8.4 Microbiological monitoring of the filling room is carried out daily for settling plate, centrifugal air sampler and swab testing. Personnel working in the area are also monitored for bioburden.

3.8.5 Nonviable particulate counts are taken on daily basis to ensure the cleanliness as per FED. STD. 209E

3.9 Gas purging if any

3.9.1 The vials are dosed with sterile powder with the aid of sterile nitrogen. Where as in the case of sterile Ceftazidime and sodium carbonate mixture filling, the empty vials is purged with sterile CO2, dosed with sterile CO2 and before stoppering once again purged with sterile CO2.

3.9.2 For media fill dosing of powder shall be done using compressed air. Don’t use other gases.

3.10 Optical testing of filled vials

3.10.1 After filling and sealing the vials are subjected to optical testing to detect for the presence of any bad seals (sealing defects), surface defects, cracks on the vial surface, presence of any distinguishable foreign particles in the powder etc., The rejected vials are collected separately and destroyed. And the good vials are allowed into the packing hall for labeling and final packing.

4.0 BRIEF PLAN FOR MEDIA FILL

The vial filling process described above is simulated for media fill trials. Sterile Lactose Powder is used for filling purpose, which is microbiologically inert. Primary packaging material like vials, rubber stopper and Equipments for washing, sterilization, filling and sealing of vials are same as used in routine operations. At least 9000 Vials are filled with 0.5 gm Lactose powder followed by 5.0 ml sterile Soybean Casein Digest Media vials are stoppered and then sealed. Different set, of primary packing material, used for vial filling, will be used for the media fill trials. There will be one media fill trial with one set of primary packaging material or theory of bracketing will be adopted. During filling, worst case situations are simulated where possible. Media before and after filling is checked for positive (Growth Promotion Test) and negative control (Sterility Test). Filled vials are incubated at 22.5 ± 2.5°C for 7 days followed by 32.5 ± 2.5 °C for further 7 days.

4.1 Procedure:

4.1.1 To simulate the worst case conditions in the filling room during media fills, the minimum number of personnel to participate during the media fill operations will be:

Filling room

Production

QA/QC

Engineering

Total

Class 100 Powder Filling Room

2

1

1

4

4.1.2 Filling and QA/QC operators must remain in the room during the fill except during changing gloves, goggles, etc.

4.1.3 Operator requirements.

4.1.3.1 Each operator working in a class 100 area will require to be certified for level I certification. The following are the minimum requirements for operators to maintain class 100 Certification.

4.1.3.2 Gowning training per CP-QC-MICRO-016.

4.1.3.3 Gowning certification per CP-QC-MICRO-008.

4.1.3.4 Operators are required to participate in at least one media fill per year for annual certification (except initial media fill).

4.1.3.5 Operators trained to perform setup of fill line must also p0erform setup of fill line for media fill.

4.1.3.6 The amount of time required for everyone to be in the class 100 rooms is 2 hours minimum.

4.1.3.7 All the employees entering the filling room will require to be monitored per CP-QC-MICRO-004.

4.1.3.8 Contrary to the regular fills, vials with high and low fills, particles, cosmetic defects, etc., will be included as part of media fills. Vials will not be discarded unless they are cracked, broken, have no cap or no stopper, or not properly filled (no media/no water). The vials are also rejected if the stoppers and caps are improperly applied and the contents of the vials have exposed to the non-sterile environment.

4.1.3.9 The duration of routine media fill runs will be the time required to fill minimum of 5000 vials at the slowest speed (20-25units per minute for all sizes of vials).

4.1.3.10 The inspection log sheet (Attachment 10) in the batch record will list the vial and box number of media filled vials.

4.1.3.11 Sanitize the fill line surfaces such as conveyor belt, and the other machine parts that are not subject to autoclave, by wiping with 70% Sterile Isopropyl Alcohol.

4.1.3.12 Growth promotion samples will be pulled randomly during the process simulation. After inoculation, the samples will be incubated in parallel with the process simulation units.

4.1.3.13 Following are the reasons to invalidate the media fill:

4.1.3.14 Failure of growth promotion test of media.

4.1.3.15 Power outage for more than 3 hours.

4.1.3.16 After the initial qualification, one (1) media fill will performed twice a year. This stipulates that there were no changes in normal production procedures and no action limits were exceeded.

4.1.3.17 FILLING LINE (Refer to Attachment 1,2 or 3 for media fill requirements.

4.1.3.18 Setup the filling equipment and components per Aurobindo Pharma standard operating procedures for filling operations.

4.1.3.19 The sterilized filling equipments and components can be held in the class 100 area for no more than three (3) days prior to fill date.

4.1.3.20 The media fill start time, break period start and end time, and media fill completion time will be logged in Attachment2.

Justification for using sterile Lactose to simulate the vial filling process

1. Flow ability of Lactose through hopper and dosing wheel is good.

2. It does not inhibit the growth of microorganisms at the selected concentration (approximately 10% w/v).

3. Lactose is soluble in media and water.

4. Could be sterilized by gamma radiation.

Reference for usage of Lactose as placebo materials are also available with “ process simulation testing for aseptically filled products” Vol. 50 No 6, Nov-Dec 1996, supplement Appendix 1 Page 5.13 which is based on PDA guideline Technical monograph no 2 validation of aseptic filling for solution drug products 1980 and Technical Report no. 6, validation of aseptic drug powder filling process 19845.0 EQUIPMENT

Following equipment are utilized during media fill trials

Name of the equipment

Tag Nos.

Linear vial washer

VWD001

Bung processor

BWD001

Tunnel sterilizer

TSD001

Sterile powder Filling machine

VFD001

Vial sealing machine

SMD001

Optical testing machine

OTD001

6.0 CONTAINER CLOSURE SYSTEMS

Vial

Stopper

Seal

5 ml Molded

20mm Grey butyl

20mm Bromo butyl

20mm Flip off

20mm Tear of

7.5 ml Molded

20mm Grey butyl

20mm Bromo butyl

20mm Flip off

20mm Tear of

10 ml molded

20mm Grey butyl

20mm Grey Bromo butyl ready for sterilization

20mm Flip off

20mm Tear off

10 ml tubular

20mm Bromo butyl

20mm Grey Bromo butyl ready for sterilization

20mm Flip off

15 ml molded

20mm Grey butyl

20mm Bromo butyl

20 mm Flip off

15ml Tubular

20mm Bromo butyl

20mm Grey Bromo butyl ready for sterilization

20mm Flip off

20 ml molded

20mm Grey butyl

20mm Bromo butyl

20mm Flip off

20 ml tubular

20mm Bromo butyl

20mm Grey Bromo butyl ready for sterilization

20mm Flip off

30 ml tubular

20mm Bromo butyl

20mm Grey Bromo butyl ready for sterilization

20mm Flip off

50 ml Molded

32mm Bromo butyl

32mm Grey Bromo butyl ready for sterilization

32mm Flip off

100 ml Molded

32mm Bromo butyl

32mm Grey Bromo butyl ready fir sterilization bags

32mm Flip off

For Media fill purpose-bracketing approach shall be adopted. Trials shall be taken with different packing system and all packing system shall be simulated over a year. In case if any new container closure system / type in included (a part from those described above) shall be simulated before commercial production of that particular size.

1st half Year :

Trial

Vial

Stopper

Seal

Minimum No of vials for fill simulation

1st

5 ml Molded

20mm Grey butyl

20mm Flip off

4500

20mm Bromo butyl

20mm Tear off

4500

2nd

10 ml Molded

20mm Grey butyl

20mm Flip off

4500

20mm Grey Bromo butyl ready for sterilization

4500

3rd

15 ml Molded

20mm Grey butyl

20mm Flip off

4500

20mm Bromo butyl

4500

4th

15 ml Tubular

20mm Bromo butyl

20mm Flip off

4500

20mm Grey Bromo butyl ready for sterilization

4500

5th

30ml Tubular

20mm Bromo butyl

20mm Flip off

4500

20mm Grey Bromo butyl ready for sterilization

4500

2nd half of Year:

Trial

Vial

Stopper

Seal

Minimum No of vials for fill simulation

1st

7.5 ml Molded

20mm Grey butyl

20mm Flip off

4500

20mm Bromo butyl

20mm Tear off

4500

2nd

10 ml Tubular

20mm Bromo butyl

20mm Flip off

4500

20mm Grey Bromo butyl ready for sterilization

4500

3rd

20 ml Molded

20mm Grey butyl

20mm Flip off

4500

20mm Bromo butyl

4500

4th

20 ml Tubular

20mm Bromo butyl

20mm Flip off

4500

20mm Grey Bromo butyl ready for sterilization

4500

5th

50 ml Molded

32mm Bromo butyl

32mm Flip off

4500

32mm Grey Bromo butyl ready for sterilization

4500

7.0 REVALIDATION FREQUENCY

Scheduled Revalidation:

Three Media fill trials to be carried out once in six month with one trial with each type of container closure system.

Unscheduled Revalidation:

Revalidation are also carried out in some special cases:

a. After maintenance of major breakdown in Filling Machine, Sealing Machine and HVAC system

b. Change in the Vial Filling Machine and Sealing machine itself or their critical parts

8.0 RESPONSIBILITIES

Validation team

Validation team consist of the key personnel from the following department

· Production

· QC

· QA

· Engineering

General responsibly of validation team

· Monitoring of protocol completeness, accuracy, technical excellence and applicability.

· Scheduling of validation.

· Conducting of validation runs.

· Data compilation and review.

· Validation reports preparation and recommendation thereafter (if required).

· Schedule of revalidation.

The Engineering Department shall be responsible for the following.

· Carrying out the repairs/modifications (if required) prior to validation runs.

The responsibility of QC - Microbiology Lab shall be as follows:

· Preparation and qualification of the microbiological growth support medium.

· Carrying out the monitoring (settling plate, air sampling, swab testing and personnel monitoring) in critical area during media fill trials.

· Inoculation and incubation of empty vials and stoppers, sterility test of lactose used for media fill trials.

· Incubation of the remaining media to confirm its sterility.

· Observation of incubated filled vials after 7 and 14 days.

· To carry out investigations in case the media fill trial fails.

· Detecting the organism in the vials showing microbial growth.

Responsibilities of Production Department are:

· Procuring sterile Lactose powder (gamma radiated).

· Providing the machine when agreed upon with the validation group.

· Providing machine operators and supervisory staff.

· Sterilization of media, empty vials, rubber stoppers and drying of rubber stoppers.

· Providing samples wherever necessary.

· Carrying out the aseptic filling process.

· Checking the damaged vials after filling and sealing.

· Incubation and ensuring security of media filled vials.

· Cleaning and sanitization of area and equipments after media fill.

· Destruction of media filled vials after evaluation and authorizations from Q.A.

9.0 B.VALIDATION METHODOLOGY

9.1 PRE-VALIDATION CHECKS

Prior to taking up the validation of aseptic filling, it should be ensured that all equipments, utilities and processes are validated and all instruments are calibrated.

It should be ensured that the aseptic filling room is at a positive pressure with respect to the surrounding areas.

Particulate count for non-viable particles (static conditions) should be within acceptable limits of environment control sop.

9.2 CHECKS DURING THE VALIDATION RUNS

9.2.1 Personnel Monitoring:

Ensure that personnel entering the sterile area have followed the gowning and entry procedures as per standard Operating Procedure and have been qualified as per standard Operating Procedure. Personnel should undergo, monitoring by RODAC plate method after completion of the trial and before leaving the sterile area for monitoring bio-burdens levels.

9.2.2 Environmental Monitoring

Environmental Monitoring of the sterile area should be done as per standard Operating Procedure

Swab test for surfaces like walls/floor etc. of the filling room and surfaces of the vial-filling machine.

Monitoring of microbial counts in air shall be done by air sampling by settling plate method.

Monitor the environmental conditions for temperature, differential pressure, relative humidity and Non-viable airborne particle counts.

The critical area should meet the specifications of class 10,000 clean room and the area under vertical laminar air flow unit should meet the specifications of class 100 clean room as per the Federal Standards 209E.

Environmental and personnel monitoring result should comply the acceptance limit as written in Standard Operating Procedure for Environmental Monitoring

9.3 VALIDATION WITH MEDIA

At least 9000 vials shall be filled for each trial run in duration of 8 hours (an additional step of filling media in the empty vials reduces the normal operating speed) to simulate the conditions.

9.4 Sterilization of Lactose powder:

Lactose powder (endotoxin free) shall be packed in Aluminum foil pouches (5 Kg per pack) wrap these packs in double polybags. These packs shall be packed in the shippers as specified by ISOMED. Each shipper is filled with two packs of 5kg Lactose and a 50 gm sample pouch is placed below the pouches for sterility testing and growth promotion test of the Lactose powder after sterilization. These shippers are then sent for sterilization by gamma radiation at ISOMED (BARC), Mumbai.

9.5 Transfer of Sterilized Lactose packs into critical area

On receipt of lactose packs after sterilization, the pack shall be vacuum cleaned. The outer carton and polybag shall be removed in the decartoning room. The shippers are opened and sample packs of the Lactose are collected and tested for sterility and growth promotion. The container shall be sanitized as per standard Operating Procedure and shall be transferred to sterile quarantine. The packs shall be stored in sterile quarantine under LAF unit. Disinfect the packs as per SOP and transfer to filling room prior to vial filling operation.

9.6 Suitability Test of lactose

a. Dissolve 500 mg sterile Lactose in 5 ml of Soybean Casein Digest media in a test tube to check solubility of Lactose in media.

b. Establish the concentration lactose, which should be non-inhibitory.

Microbiology Department shall maintain the record of suitability study

9.7 Preparation of Media :

Dissolve 600.0 gm of SCDM powder in 20 L. of WFI (DW) in SS carboys and distribute in three carboy i.e. approximately 6.5L liters/carboy (Concentration of media 30 gm SCDM/lit). Close the Carboys. Sterilize the carboys at validated sterilization parameter. Unload the media in critical area. Transfer the Media Carboy to Powder Filling Room as per SOP. Remove 800 ml media aseptically from each trial for Growth Promotion Test.

Note :

A) Store the sterilized carboys filled with media under LAF till it is used. The media preparation and sterilization will be done in presence of Microbiologist.

B) Sterilized media should be used for media fill trial only after it passes in GPT and shows no contamination when stored for 7 days at room temperature.

9.8 Method of Growth Promotion test

Objective of the test: To ensure that media used for the sterility test is capable of microbial growth promotion

a. Aseptically withdraw 800 ml of autoclaved media and aseptically transfer 100 ml each in 8 sterilized tubes.

b. Out of the 8 tubes, 2 tubes are inoculated with about 10 – 100 cells of Candida albicans ATCC 10231, 2 tubes with about 10 – 100 cells of Bacillus subtillis ATCC 6633 and 2 tubes with about 10 – 100 cells of Aspergillus niger ATCC 16404. The Inoculum for growth promotion test are prepared as per the SOP.

c. Incubate the tubes inoculated with Bacillus subtillis at 32.5 ± 2.5°C for maximum 3 days and incubate the tubes inoculated with Candida albicans and Aspergillus Niger at 22.5 ± 2.5°C for maximum 5 days.

d. Incubate 2 tubes (Un-inoculated) of SCDM at 22.5 ± 2.5°C for 7 days followed by 7 days at 32.5 ± 2.5°C as Negative Control.

Acceptance Criteria: Media inoculated with microorganism should promote growth of microbes and un-inoculated media should remain sterile.

9.9 Component Preparation

Components i.e. Vials, machine parts are washed. Rubber Stoppers are sterilized, dried and transferred to vial filling room. Following SOPs are applicable.

Operation

SOP No

Washing of Rubber stopper

Steam Sterilization and Drying of Rubber stopper

Cleaning of vial filling machine parts

Steam Sterilization of Filling Machine Parts

Washing of Vials

Tunnel Sterilization of Vials

9.10 Media Fill Operations

a. Obtain the line clearance before starting filling operation of the batch as per Standard Operating Procedure for any left over Raw Material, Vials, Rubber stoppers of previous batch.

b. Check the area and machine for cleanliness.

c. Check the temperature, Differential Pressure and Relative Humidity of Powder filling Room.

d. Transfer carboy-containing media to filling room. Connect volumetric liquid filling assembly with the carboy

e. Transfer the sterilized rubber stopper, Lactose packs and machine parts to powder filling room and assemble the machine parts

f. Check and ensure that all media filled carboy containers are free from microbial growth / turbidity.

g. Take 10 vials in a sterile conical flask containing 500 ml SCD media. Incubate the media at 22.5 ± 2.5°C for 7 days followed by further 7 days at 32.5 ± 2.5°C. Check for any microbial growth in the flasks.

h. Adjust the powder dosing to deliver 0.5 g of Lactose /vial.

i. Adjust the liquid filling machine to deliver 5.0 ml media in each vial.

j. Run the machine to fill only media for checking of volume variation. Approximately 50 vials may require for volume adjustment and checking. These vials will be sealed and kept separately for investigation purpose if required in case of failure.

k. Run the machine to fill only Lactose for checking of weight variation. Approximately 200 vials may require for weight adjustment and checking. These vials will be sealed and kept separately for investigation purpose if required in case of failure.

l. Record three consecutive set of observations of weight and volume checking from all filling station

m. Operate the Filling Machine and Turn Tables to fill Lactose followed by liquid media in Vials

n. Seal filled and stoppered vials

o. Following sequence will be followed for normal operation:

p. Vials from Tunnel Outlet ( TurnTable ( sterilized Lactose powder ( Media fill( Stoppering ( Sealing ( Visual inspection ( Invert the vials ( Incubation.

q. Collect these media and Lactose filled, stoppered and sealed vials in SS trays identify the trays with number and time of filling. Invert the vial so that the media comes in contact with all the inner surfaces of vial and stoppers.

r. Sample 30 vials for growth promotion test.

s. Fill at least 5000 vials excluding weight/volume variation check vials with one set of container closure system (10000 vials per trial). After completion of filling obtain line clearance from In Process QA chemist.

t. Operate the machine at minimum, maximum and normal production speed.

u. Requirement of Raw material and packing material:

i. Sterile lactose: 5.1 Kg

ii. Vials: 10200

iii. Stopper: 10200

iv. Seal: 10200

9.11 Growth Promotion Test of Lactose Filled Vials

Objective of the Test: To ensure that lactose filled in the sterile glass vials and sealed by rubber stopper and aluminum seal, does not inhibit the growth of microorganism

a. Collect 30 filled and sealed vials

b. 5 vials are inoculated with about 10 - 100 cells of Candida albicans ATCC 10231, 5 vials are inoculated with about 10 - 100 cells of Bacillus subtillis ATCC 6633, 5 vials are inoculated with about 10 - 100 cells of Aspergillus niger ATCC 16404 and five vials each of 3 environmental isolates are inoculated with about 10-100 cells. The innoculum for growth promotion test are prepared

c. Incubate the vials inoculated with Bacillus subtillis at 32.5 ± 2.5°C for maximum 3 days incubate the vials inoculated with Candida albicans and Aspergillus niger at 22.5 ± 2.5°C for maximum 5 days and incubate the environmental isolates as per their respective growth conditions.

Acceptance Criteria: Media should promote the growth within the stipulated period.

9.12 Worst case consideration:

Following conditions will be simulated to have worst case condition during the trials (indicated against the step):

1 To conduct media fill trial simulating change of personnel, tea break, lunch break etc.

2 Deliberately switch off the HVAC system and LAFs in critical area ( Cooling & Filling Zone) for 5 minutes in any one of the trials

3 Simulate the interlocking failure by switch off the interlocking system for 30 minutes during filling operation (in any one of trials).

4 Ask one maintenance person (qualified to enter in sterile area) to enter into filling zone with his toolbox and to stay for 4 Hrs.

5 One microbiologist to enter the area for monitoring purpose during media fill trials.

6 To extend the filling time from 8 Hrs to 16 Hrs in any one of the three trials.

7 Intervene the filling operation by simulating the following operation

a) Adjustment of the fill weight

b) Charging and recharging of the Lactose in hopper

c) Charging and recharging of rubber stopper in hopper

d) Adjustment of the sensors if required or touch the sensors

e) Set the rubber stopper chute

f) Setting of Nitrogen dosing and setting or Carbon Dioxide Purging (if applicable)

g) Adjustment of roller for pressing of stopper

h) Removal of choked piston

i) Removal of dropper filled and unfilled vials from turntable

j) Cleaning of conveyor on dropping of filled vials on conveyor (deliberately simulate the operation by stopping the filling operation and pouring the content of vial on the conveyor

k) Open the entry door of powder filling room so that, differential pressure of the room goes below standard differential pressure

l) Stop the sealing machine and sterilizing tunnel when the turntable of filled and unfilled vials is full. Stop the filling operation. Let the turntable rotate for 30 minutes

m) Ask maintenance person to simulate minor maintenance job.

9.13 INTERVENTIONS

9.14 The following interventions will be performed throughout the process of the fill. All interventions must be completed prior to the end of the fill.

9.14.1 Change the count on the dosing wheel to adjust the weight of the powder.

9.14.2 Adjust sensors.

9.14.3 Adjust the stopper arm for height.

9.14.4 Remove the broken glass vials from the fill line.

9.14.5 Perform a clean up of a powder spill using a vacuum cleaner.

9.14.6 Open safety panels on the filling machine. Keep the panels open for 10 minutes during the machine stoppage.

9.14.7 Hold the filling room door open for 60 seconds.

9.14.8 Shut the filling line down for a minimum of 1 hour, but do not clear the in-feed table. This operation can be coordinated at the break time.

9.14.9 Open the swing-open conveyor to get behind the machine and spend 5 minutes simulating checking the pressures on the Magnehelic Gauges.

9.14.10 Cause the conveyor jam at in feed and out feed and unjam the conveyor.

9.14.11 Cause the vial jam and unjam at the star wheel.

9.14.12 Record actual or unexpected interventions(s) on

9.14.13 Attachment 2,3 or 4.

9.15 BREAK PERIOD

9.15.1 The machines will be stopped during the break period will be of 1 hour duration. This time includes time needed for gowning, degowning and actual break time. The interventions outlined in section 5.13.3 may be timed properly to coincide with the scheduled break. Any operational malfunctions experienced before the simulation can be considered to satisfy the simulation requirements. When logging the events, indicate if the intervention was actual or simulated.

9.15.2 Run the machine in the slowest possible production speed to simulate the worst-case condition. Maintain the same speed (20-25 vials per minute) through out the fill period. Log the actual line speed setting used at this time.

9.15.3 Vials will be removed from the fill line if the following occur:

9.15.3.1 Vials with no stoppers or defective stopper.

9.15.3.2 Vials with no powder.

9.15.3.3 Vials with no water.

9.15.3.4 Tipped vials.

9.15.3.5 Vials with no seals or defective seals.

9.15.3.6 The vials removed from the fill line inside the clean rooms will be discarded.

9.15.4 After the sealing operation, shake the vials to facilitate the dissolu8tion of the media in water. Shake or vortex till a clear solution is obtained. Invert each vial to let the media solution come in contact with all sides of the vial including the stopper. Examine each vial for clarity, glass defects, proper placements of stopper and seal. Reject the vials found defective. Number the inspected vials with marking pen with sequential numbers starting from one (1) after inspection and arrange the vials in boxes. Label the boxes with lot number, number of vials inside the box, fill date and sequential vial numbers from beginning to end contained in the box. Deliver the boxes to Microbiology laboratory for incubation.

9.15.5 After each fill, if necessary. Remove dosing wheel, water pump, and water carboy and stopper bowl and sterilize for the next usage.

9.15.6 The media filled containers will be incubated for two 7-day periods at 20-25oC.

9.15.7 Visual inspection will be performed during incubation period on the last day of each 7-day period. Inspection will be performed by QA/QC personnel and will be recorded at Attachment 1 on CP-QC-MICRO-021.

9.15.8 For all media fills, refer to Attachment 1,2 or 3 for validation/QA requirements.

9.16 Personnel Involvement:

During Media filling operation minimum 04 and maximum 06 number of persons will be present in side the filling room. All persons involved in routine aseptic operation should be included in any one of the following critical aseptic operations in the trial.

a. Unloading of stopper from steam Sterilizer

b. Unloading of machine parts from steam Sterilizer

c. Charging of RM and rubber stoppers into hopper

d. Vial filling machine assembling and setting

e. Vial filling operation.

All person involved in routine filling operation should involve in media filling operation over a period of 1 year

One Microbiologist (QC), qualified to enter into critical area, will enter into critical area for area monitoring, personnel monitoring and sampling. All microbiologists involved in sampling and environmental monitoring during normal manufacturing periods will take part in system simulation run over a period of one year.

The maintenance personnel, qualified to enter into critical area, shall be required to enter the critical area and to remain in the area for at least 4 hrs. Duration with the tool box and will be monitored for personnel counts in all the trial runs. (The maintenance person will follow the entry & exit procedures as per normal production runs, but will not interfere in the proceedings during the system simulation trials).

Area of Operation

No. of Persons

Duration in Aseptic Area

Remarks

Manufacturing

04

04 Hrs x 2 Times

Come out for Lunch after 4 hrs and will go in aseptic area again after changing garments

Microbiologist [QC]

01

04 Hrs x 2 Times

Wear fresh garments for each entry. To be present during filling operation.

Supervisor

01

02 Hrs x 2 Times

Wear fresh garments for each entry

OR

Maintenance

01

04 Hrs x 1 Time

Will enter with toolbox. To be present during filling operation.

9.17 Incubation of Vials

9.17.1 Incubation at 22.5 ± 2.5°C

· Transfer the properly identified SS trays containing media filled vials into the Incubation room.

· Maintain the room temperature at 22.5 ± 2.5°C for 7 days.

· After 7 days of incubation at 22.5 ± 2.5°C observe each vial visually for any type of growth/Turbidity.

· Identify the vials with growth/turbidity (if any) and send these to Microbiology laboratory for further investigations.

9.17.2 Incubation at 32.5 ± 2.5°C

· Maintain the temperature of incubation room at 32.5 ± 2.5°C for next 7 days.

· After 7 days of incubation at 32.5 ± 2.5°C observe each vial visually for any type of growth/Turbidity

· Identify the vials with growth/turbidity (if any) and send these to Microbiology laboratory for further investigations.

9.18 Growth Promotion Test of Lactose Filled Vials after 14 days Incubation

Objective of the Test: To ensure that Lactose filled in the sterile glass vials and sealed by rubber stopper and aluminium seal, does not inhibit the growth of microorganism after incubation of 14 days.

a. Collect 30 filled and sealed vials

b. 5 vials are inoculated with about 10 - 100 cells of Candida albicans ATCC 10231, 5 vials are inoculated with about 10 - 100 cells of Bacillus subtillis ATCC 6633, 5 vials are inoculated with about 10 - 100 cells of Aspergillus niger ATCC 16404 and five vials each of 3 environmental isolates are inoculated with about 10-100 cells. The inoculums for growth promotion test are prepared.

d. Incubate the vials inoculated with Bacillus subtillis at 32.5 ± 2.5°C for maximum 3 days and incubate the vials inoculated with Candida albicans and Aspergillus niger at 22.5 ± 2.5°C for maximum 5 days and incubate the environmental isolates as per their respective growth conditions.

Acceptance Criteria: Media should promote the growth within stipulated period.

9.19 Destruction of Media filled Vials after 14 days Incubation

Media filled Vials are steam sterilized and destroyed by incineration after the 14 days incubation and observation.

C. ACCEPTANCE CRITERIA:

9.20 ACCEPTANCE CRITERIA:

9.20.1 An acceptable SCDM (or TSB) media fill run will consist of a minimum of 5000 units filled with sterile SCDM (or TSB) powder followed by sterile Water For Injection.

9.20.2 The duration of each media fill run will be the time required to fill minimum of 5000 vials of each size at the lowest speed of the machine (20-25 vials per minute).

9.20.3 Requirements listed in Attachment 1,2 or 3 have been met as appropriate.

9.20.4 Positive-control units filled with SCDM (or TSB) must exhibit growth of the specified growth promotion organisms at the specified incubation parameters per CP-QC-MICRO-015.

9.20.5 Ideally the contamination rate should be zero. However, the FDA guidelines suggest the contamination rate of less than 0.1% with a 95% confidence level. Initial qualification requires 3 successful consecutive media fill tests. During the initial qualification, if ther are two (2) contaminated units in a single run, or one (1) each in 2 run, the qualification is stopped, cause is investigated and 3 media fill batches are repeated.

If the level of contamination exceeds 01 vial per 9000 vials, then cause of contamination should be investigated and one more media fill run should be conducted. If the number of contaminated units exceeds 03 per 9000 vials, investigate the reasons of failure, rectify and then repeat the trials.

The aseptic drug powder filling process shall be considered validated if all media fill runs are completed successfully.

D.ACTION TAKEN IN CASE OF SYSTEM SIMULATION FAILURES

In the event of any one or more trial results out of three trials fails in the test for sterility, before starting fresh product campaign, following actions will be taken.

Characterisation of microorganisms up to species level, shall be carried out to find out the source of contamination.

Review of following records for 60 days prior to system simulation trial failure after the failing batch (Lactose trial) processing is carried out:

In addition to this following observations will be closely controlled and monitored for subsequent 60 days.

a.Environmental records for manufacturing and testing area for temperature, relative humidity, differential pressure and non-viable particulate counts.

b.Microbiological monitoring, reports of manufacturing and testing area for settling plate, air sampling, surface monitoring and personnel monitoring.

c.Sterilization records for garments, filters, vials, stopper, machine parts, media etc.

d.Filter integrity record of nitrogen, Carbon Dioxide and air supply filters.

e.WFI monitoring records.

f.Cleaning and sanitizing records.

g.Batch records of system simulation trials to find out the sign of any failures or anomalies.

h.All other points mentioned in SOP for investigation will be included for investigation.

Based on the above review, the most likely cause of failure will be identified and an action plan will be made to avoid such occurrence in future.

The area will be monitored for bio-burden and after getting satisfactory area monitoring reports for 3 consecutive days; three trials with Lactose will be taken.

All three such trials should pass as per the acceptance criteria for sterility.

Note: No commercial production batches will be produced till media fill runs pass as per acceptance criteria.

1.1.1 CORRECXTIVE ACTIONS:

1.1.1.1 If any positive unit (s) is/are identified such that an alert or action level is reached, an investigation will be performed and documented per CP-QC-MICRO-009.

1.1.1.2 The investigation will include but not be limited to the following:

1.1.1.2.1 Microbial environmental monitoring data.

1.1.1.2.2 Particulate monitoring data.

1.1.1.2.3 Bio burden data (Prior to pre-filtration and prior to final filtration).

1.1.1.2.4 Personnel monitoring data (finger impressions, etc.,).

1.1.1.2.5 Sterilization for media, equipment and commodities.

1.1.1.2.6 HEPA filter evaluation (airborne particle levels, smoke challenge testing, velocity measurements, etc).

1.1.1.2.7 Room airflow patterns and pressures.

1.1.1.2.8 Operator techniques and training.

1.1.1.2.9 Unusual events that occurred during the media-fill.

1.1.1.2.10 Storage conditions of sterile commodities.

1.1.1.2.11 Identification of contaminants.

1.1.1.2.12 House keeping procedures and training.

1.1.1.2.13 Calibration of sterilization equipment.

1.1.1.2.14 Pre- post- filter integrity test data.

1.1.1.2.15 Product and/or process defects, and/or limitation of inspection process.

1.1.1.2.16 Documented disqualification of samples for obvious reasons prior to final reading.

1.1.1.3 When action levels are exceeded: The investigation will include a prompt review of all appropriate records relating to the aseptic production lots between the current media fill and the last successful media fill. The filling room will immediately placed out of service and an “Out of service” tag will be placed by the entrance of the filling room. A written notification will be distributed by quality assurance to management.

1.1.1.4 Alert and action level: The number of verified positives should be less than 3 contaminated vials per fill (5000 vials, with contamination rate of less than 0.1% with a 95% confidence level).

1.1.2 When action and alert levels are exceeded, routine production will not be resumed until acceptable media test run results are achieved.

E.DOCUMENTATION

The following documents shall be maintained in one binder.

· Summary report of Media fill trial

· Approved Validation protocol.

· Executed Batch Production Record for System Simulation Test ( Media Fill Trials).

· Environmental and personnel monitoring report of the critical area during the trials.

· Analysis of experimental results of the trials.

· Investigation report and discussion for the cause in case of failure trial (i.e. not meeting the acceptance criteria).

· Investigation report for microbiological identification if growth is observed.

· Recommendation for the corrective action [if any] required.

· Summary and Conclusion duly approved by Head of the Quality Assurance Department.

The observations during the system simulation test (Media fill trials) should be recorded in the System Simulation Test record and its attachments .

F. Conclusion:

Acceptable result of all media run will establish the suitability of Standard Operating Procedure for area cleaning and disinfection, cleaning and sterilization of component, aseptic transfer of component, raw material and primary packaging material, Environmental control system for temperature, Relative Humidity, Differential pressure and viable / nonviable particulate load and finally the training and practices of personnel.