Aseptic Processing & Media Fill

ASEPTIC PROCESSING & MEDIA FILL TEST
1
Discussed & Presented By: Muhammad Luqman Ikram

ASEPTIC PROCESSING - OVERVIEW
• CERTAIN PHARMACEUTICAL PRODUCTS MUST BE STERILE
• INJECTIONS, OPHTHALMIC PREPARATIONS, IRRIGATIONS
SOLUTIONS, HAEMODIALYSIS SOLUTIONS
• TWO CATEGORIES OF STERILE PRODUCTS
• THOSE THAT CAN BE STERILIZED IN FINAL CONTAINER
(TERMINALLY STERILIZED)
• THOSE THAT CANNOT BE TERMINALLY STERILIZED AND MUST BE
ASEPTICALLY PREPARED
2Discussed & Presented By: Muhammad Luqman Ikram

ASEPTIC PROCESSING
• OBJECTIVE IS TO MAINTAIN THE STERILITY OF A PRODUCT,
ASSEMBLED FROM STERILE COMPONENTS
• OPERATING CONDITIONS SO AS TO PREVENT MICROBIAL
CONTAMINATION

OBJECTIVE
• TO REVIEW SPECIFIC ISSUES RELATING TO THE MANUFACTURE OF
ASEPTICALLY PREPARED PRODUCTS:
• MANUFACTURING ENVIRONMENT
• CLEAN AREAS
• PERSONNEL
• PREPARATION AND FILTRATION OF SOLUTIONS
• PRE-FILTRATION BIOBURDEN
• FILTER INTEGRITY/VALIDATION
• EQUIPMENT/CONTAINER PREPARATION AND STERILIZATION
• FILLING PROCESS
• VALIDATION OF ASEPTIC PROCESSES

MANUFACTURING ENVIRONMENT
CLASSIFICATION OF CLEAN AREAS
• COMPARISON OF CLASSIFICATIONS
5
WHO GMP US 209E US Customary ISO/TC (209)
ISO 14644
EEC GMP
Grade A M 3.5 Class 100 ISO 5 Grade A
Grade B M 3.5 Class 100 ISO 5 Grade B
Grade C M 5.5 Class 10 000 ISO 7 Grade C
Grade D M 6.5 Class 100 000 ISO 8 Grade D
Table 1

CLASSIFICATION OF CLEAN AREAS
• CLASSIFIED IN TERMS OF AIRBORNE PARTICLES (TABLE 2)
6
Grade At rest In operation
maximum permitted number of particles/m3
0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm > 5 µ
A 3 500 0 3 500 0
B 3 500 0 350 000 2 000
C 350 000 2 000 3 500 000 20 000
D 3 500 000 20 000 not defined not defined
“At rest” - production equipment installed and operating
“In operation” - Installed equipment functioning in defined
operating mode and specified number of personnel present

FOUR GRADES OF CLEAN AREAS:
• GRADE D (EQUIVALENT TO CLASS 100,000, ISO 8):
• CLEAN AREA FOR CARRYING OUT LESS CRITICAL STAGES IN MANUFACTURE
OF ASEPTICALLY PREPARED PRODUCTS EG. HANDLING OF COMPONENTS
AFTER WASHING.
• GRADE C (EQUIVALENT TO CLASS 10,000, ISO 7):
• CLEAN AREA FOR CARRYING OUT LESS CRITICAL STAGES IN MANUFACTURE
OF ASEPTICALLY PREPARED PRODUCTS EG. PREPARATION OF SOLUTIONS
TO BE FILTERED.
• GRADE B (EQUIVALENT TO CLASS 100, ISO 5):
• BACKGROUND ENVIRONMENT FOR GRADE A ZONE, EG. CLEANROOM IN
WHICH LAMINAR FLOW WORKSTATION IS HOUSED.

• GRADE A (EQUIVALENT TO CLASS 100 (US FEDERAL STANDARD 209E),
ISO 5 (ISO 14644-1):
• LOCAL ZONE FOR HIGH RISK OPERATIONS EG. PRODUCT FILLING,
STOPPER BOWLS, OPEN VIALS, HANDLING STERILE MATERIALS, ASEPTIC
CONNECTIONS, TRANSFER OF PARTIALLY STOPPERED CONTAINERS TO BE
LYOPHILIZED.
• CONDITIONS USUALLY PROVIDED BY LAMINAR AIR FLOW
WORKSTATION.
• EACH GRADE OF CLEANROOM HAS SPECIFICATIONS FOR VIABLE AND
NON-VIABLE PARTICLES
• NON-VIABLE PARTICLES ARE DEFINED BY THE AIR CLASSIFICATION (SEE
TABLE 2)

• LIMITS FOR VIABLE PARTICLES (MICROBIOLOGICAL CONTAMINATION)
9
Grade Air sample
(CFU/m3)
Settle plates (90mm
diameter)
(CFU/4hours)
Contact plates
(55mm
diameter)
(CFU/plate)
Glove print
(5 fingers)
(CFU/glove)
A < 1 < 1 < 1 < 1
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -
Table 3
– These are average values
– Individual settle plates may be exposed for 4 hours
• Values are for guidance only - not intended to represent specifications
• Levels (limits) of detection of microbiological contamination should be
established for alert and action purposes and for monitoring trends of air
quality in the facility

SOURCE OF CONTAMINATION

ENVIRONMENTAL MONITORING
• PHYSICAL
• PARTICULATE MATTER
• DIFFERENTIAL PRESSURES
• AIR CHANGES, AIRFLOW PATTERNS
• CLEAN UP TIME/RECOVERY
• TEMPERATURE AND RELATIVE HUMIDITY
• AIRFLOW VELOCITY

ENVIRONMENTAL MONITORING - PHYSICAL
• PARTICULATE MATTER
• PARTICLES SIGNIFICANT BECAUSE THEY CAN CONTAMINATE
AND ALSO CARRY ORGANISMS
• CRITICAL ENVIRONMENT SHOULD BE MEASURED NOT MORE
THAN 30CM FROM WORKSITE, WITHIN AIRFLOW AND DURING
FILLING/CLOSING OPERATIONS
• PREFERABLY A REMOTE PROBE THAT MONITORS CONTINUOUSLY
• DIFFICULTIES WHEN PROCESS ITSELF GENERATES PARTICLES (E.G.
POWDER FILLING)
• APPROPRIATE ALERT AND ACTION LIMITS SHOULD BE SET AND
CORRECTIVE ACTIONS DEFINED IF LIMITS EXCEEDED

• DIFFERENTIAL PRESSURES
• POSITIVE PRESSURE DIFFERENTIAL OF 10-15 PASCALS SHOULD BE
MAINTAINED BETWEEN ADJACENT ROOMS OF DIFFERENT
CLASSIFICATION (WITH DOOR CLOSED)
• MOST CRITICAL AREA SHOULD HAVE THE HIGHEST PRESSURE
• PRESSURES SHOULD BE CONTINUOUSLY MONITORED AND
FREQUENTLY RECORDED.
• ALARMS SHOULD SOUND IF PRESSURES DEVIATE
• ANY DEVIATIONS SHOULD BE INVESTIGATED AND EFFECT ON
ENVIRONMENTAL QUALITY DETERMINED

• AIR CHANGES/AIRFLOW PATTERNS
• AIR FLOW OVER CRITICAL AREAS SHOULD BE UNI-DIRECTIONAL (LAMINAR FLOW)
AT A VELOCITY SUFFICIENT TO SWEEP PARTICLES AWAY FROM FILLING/CLOSING
AREA
• FOR B, C AND D ROOMS AT LEAST 20 CHANGES PER HOUR ARE USUSALLY
REQUIRED
• CLEAN UP TIME/RECOVERY
• PARTICULATE LEVELS FOR THE GRADE A “AT REST” STATE SHOULD BE ACHIEVED
AFTER A SHORT “CLEAN-UP” PERIOD OF 20 MINUTES AFTER COMPLETION OF
OPERATIONS (GUIDANCE VALUE)
• PARTICLE COUNTS FOR GRADE A “IN OPERATION” STATE SHOULD BE MAINTAINED
WHENEVER PRODUCT OR OPEN CONTAINER IS EXPOSED

• TEMPERATURE AND RELATIVE HUMIDITY
• AMBIENT TEMPERATURE AND HUMIDITY SHOULD NOT BE
UNCOMFORTABLY HIGH (COULD CAUSE OPERATORS TO
GENERATE PARTICLES) (18°C)
• AIRFLOW VELOCITY
• LAMINAR AIRFLOW WORKSTATION AIR SPEED OF APPROX
0.45M/S ± 20% AT WORKING POSITION (GUIDANCE
VALUE)

PERSONNEL
• MINIMUM NUMBER OF PERSONNEL IN CLEAN AREA
• ESPECIALLY DURING ASEPTIC PROCESSING
• INSPECTIONS AND CONTROLS FROM OUTSIDE
• TRAINING TO ALL INCLUDING CLEANING & MAINTENANCE STAFF
• INITIAL AND REGULAR
• MANUFACTURING, HYGIENE, MICROBIOLOGY
• SHOULD BE FORMALLY VALIDATED AND AUTHORIZED TO ENTER ASEPTIC
AREA
• SPECIAL CASES
• SUPERVISION IN CASE OF OUTSIDE STAFF
• DECONTAMINATION PROCEDURES (E.G. STAFF WHO WORKED WITH
ANIMAL TISSUE MATERIALS)

PERSONNEL (2)
• HIGH STANDARDS OF HYGIENE AND CLEANLINESS
• SHOULD NOT ENTER CLEAN ROOMS IF ILL OR WITH OPEN
WOUNDS
• PERIODIC HEALTH CHECKS
• NO SHEDDING OF PARTICLES, MOVEMENT SLOW AND
CONTROLLED
• NO INTRODUCTION OF MICROBIOLOGICAL HAZARDS
• NO OUTDOOR CLOTHING BROUGHT INTO CLEAN AREAS,
SHOULD BE CLAD IN FACTORY CLOTHING
• CHANGING AND WASHING PROCEDURE
• NO WATCHES, JEWELLERY AND COSMETICS
• EYE CHECKS IF INVOLVED IN VISUAL INSPECTION

PERSONNEL (3)
• CLOTHING OF APPROPRIATE QUALITY:
• GRADE D
• HAIR, BEARD, MOUSTACHE COVERED
• PROTECTIVE CLOTHING AND SHOES
• GRADE C
• HAIR, BEARD, MOUSTACHE COVERED
• SINGLE OR 2-PIECE SUIT (COVERING WRISTS, HIGH NECK),
SHOES/OVERSHOES
• NO FIBRES/PARTICLES TO BE SHED
• GRADE A AND B
• HEADGEAR, BEARD AND MOUSTACHE COVERED, MASKS, GLOVES
• NOT SHEDDING FIBRES, AND RETAIN PARTICLES SHED BY OPERATORS

PERSONNEL (4)
• OUTDOOR CLOTHING NOT IN CHANGE ROOMS LEADING TO
GRADE B AND C ROOMS
• CHANGE AT EVERY WORKING SESSION, OR ONCE A DAY (IF
SUPPORTIVE DATA)
• CHANGE GLOVES AND MASKS AT EVERY WORKING SESSION
• FREQUENT DISINFECTION OF GLOVES DURING OPERATIONS
• WASHING OF GARMENTS – SEPARATE LAUNDRY FACILITY
• NO DAMAGE, AND ACCORDING TO VALIDATED PROCEDURES
(WASHING AND STERILIZATION)
• REGULAR MICROBIOLOGICAL MONITORING OF OPERATORS

ASEPTIC PROCESSING
• IN ASEPTIC PROCESSING, EACH COMPONENT IS INDIVIDUALLY
STERILISED, OR SEVERAL COMPONENTS ARE COMBINED WITH THE
RESULTING MIXTURE STERILIZED.
• MOST COMMON IS PREPARATION OF A SOLUTION WHICH IS FILTERED
THROUGH A STERILIZING FILTER THEN FILLED INTO STERILE CONTAINERS
(E.G ACTIVE AND EXCIPIENTS DISSOLVED IN WATER FOR INJECTION)
• MAY INVOLVE ASEPTIC COMPOUNDING OF PREVIOUSLY STERILIZED
COMPONENTS WHICH IS FILLED INTO STERILE CONTAINERS
• MAY INVOLVE FILLING OF PREVIOUSLY STERILIZED POWDER
• STERILIZED BY DRY HEAT/IRRADIATION
• PRODUCED FROM A STERILE FILTERED SOLUTION WHICH IS THEN ASEPTICALLY
CRYSTALLIZED AND PRECIPITATED
• REQUIRES MORE HANDLING AND MANIPULATION WITH HIGHER POTENTIAL FOR
CONTAMINATION DURING PROCESSING

ASEPTIC PROCESSING
PREPARATION AND FILTRATION OF SOLUTIONS
• SOLUTIONS TO BE STERILE FILTERED PREPARED IN A GRADE C
ENVIRONMENT
• IF NOT TO BE FILTERED, PREPARATION SHOULD BE PREPARED IN A
GRADE A ENVIRONMENT WITH GRADE B BACKGROUND (E.G.
OINTMENTS, CREAMS, SUSPENSIONS AND EMULSIONS)
• PREPARED SOLUTIONS FILTERED THROUGH A STERILE 0.22ΜM (OR
LESS) MEMBRANE FILTER INTO A PREVIOUSLY STERILIZED CONTAINER
• FILTERS REMOVE BACTERIA AND MOULDS
• FILTRATION SHOULD BE CARRIED OUT UNDER POSITIVE PRESSURE

ASEPTIC PROCESSING
PREPARATION AND FILTRATION OF SOLUTIONS
• TIME LIMITS SHOULD BE ESTABLISHED FOR EACH PHASE OF
PROCESSING, E.G.
• MAXIMUM PERIOD BETWEEN START OF BULK PRODUCT
COMPOUNDING AND STERILIZATION (FILTRATION)
• MAXIMUM PERMITTED HOLDING TIME OF BULK IF HELD AFTER
FILTRATION PRIOR TO FILLING
• PRODUCT EXPOSURE ON PROCESSING LINE
• STORAGE OF STERILIZED CONTAINERS/COMPONENTS
• TOTAL TIME FOR PRODUCT FILTRATION TO PREVENT ORGANISMS
FROM PENETRATING FILTER
• MAXIMUM TIME FOR UPSTREAM FILTERS USED FOR CLARIFICATION
OR PARTICLE REMOVAL (CAN SUPPORT MICROBIAL ATTACHMENT)

ASEPTIC PROCESSING
FILTER INTEGRITY
• FILTERS OF 0.22ΜM OR LESS SHOULD BE USED FOR FILTRATION OF
LIQUIDS AND GASSES (IF APPLICABLE)
• FILTERS FOR GASSES THAT MAY BE USED FOR PURGING OR
OVERLAYING OF FILLED CONTAINERS OR TO RELEASE VACUUM IN
FILTER INTERGRITY SHOULD BE VERIFIED BEFORE FILTRATION AND
CONFIRMED AFTER FILTRATION
• BUBBLE POINT
• PRESSURE HOLD
• METHODS ARE DEFINED BY FILTER MANUFACTURERS AND LIMITS
DETERMINED DURING FILTER VALIDATION

ASEPTIC PROCESSING
EQUIPMENT/CONTAINER PREPARATION & STERILIZATION
• ALL EQUIPMENT AND PRODUCT CONTAINERS SHOULD BE STERILIZED USING
VALIDATED CYCLES
• PARTICULAR ATTENTION TO STOPPERS - SHOULD NOT BE TIGHTLY PACKED AS
MAY CLUMP TOGETHER AND AFFECT AIR REMOVAL DURING VACUUM STAGE OF
STERILIZATION PROCESS
• EQUIPMENT WRAPPED AND LOADED TO FACILITATE AIR REMOVAL
• PARTICULAR ATTENTION TO FILTERS, HOUSINGS AND TUBING
• HEAT TUNNELS OFTEN USED FOR STERILIZATION/DEPYROGENATION OF GLASS VIALS
• USUALLY HIGH TEMPERATURE FOR SHORT PERIOD OF TIME 320 FOR 5MIN
• NEED TO CONSIDER SPEED OF CONVEYOR
• VALIDATION OF DEPYROGENATION (3 LOGS ENDOTOXIN UNITS)
• WORST CASE LOCATIONS
• TUNNEL SUPPLIED WITH HEPA FILTERED AIR (CLASS 100)

ASEPTIC PROCESSING
EQUIPMENT/CONTAINER PREPARATION & STERILIZATION
• EQUIPMENT SHOULD BE DESIGNED TO BE EASILY ASSEMBLED AND DISASSEMBLED,
CLEANED, SANITISED AND/OR STERILIZED
• EQUIPMENT SHOULD BE APPROPRIATELY CLEANED - O-RINGS AND GASKETS
SHOULD BE REMOVED TO PREVENT BUILD UP OF DIRT OR RESIDUES
• RINSE WATER SHOULD BE WFI GRADE
• EQUIPMENT SHOULD BE LEFT DRY UNLESS STERILIZED IMMEDIATELY AFTER CLEANING
(TO PREVENT BUILD UP OF PYROGENS)
• WASHING OF GLASS CONTAINERS AND RUBBER STOPPERS SHOULD BE VALIDATED
FOR ENDOTOXIN REMOVAL
• SHOULD BE DEFINED STORAGE PERIOD BETWEEN STERILIZATION AND USE (PERIOD
SHOULD BE JUSTIFIED)

ASEPTIC PROCESSING
PROCESS VALIDATION (MEDIA FILL TEST)
• NOT POSSIBLE TO DEFINE A STERILITY ASSURANCE LEVEL FOR
ASEPTIC PROCESSING
• PROCESS IS VALIDATED BY SIMULATING THE
MANUFACTURING PROCESS USING MICROBIOLOGICAL
GROWTH MEDIUM (MEDIA FILL)
• PROCESS SIMULATION INCLUDES FORMULATION (COMPOUNDING),
FILTRATION AND FILLING WITH SUITABLE MEDIA USING THE SAME
PROCESSES INVOLVED IN MANUFACTURE OF THE PRODUCT
• PROCESS SIMULATION FOR FORMULATION STAGE SHOULD BE PERFORMED AT
LEAST TWICE PER YEAR.

PROCESS VALIDATION (MEDIA MILL TEST)
• MEDIA FILL PROGRAM SHOULD INCLUDE WORST CASE ACTIVITIES
• FACTORS ASSOCIATED WITH LONGEST PERMITTED RUN (E.G. OPERATOR FATIGUE)
• REPRESENTATIVE NUMBER, TYPE, AND COMPLEXITY OF NORMAL INTERVENTIONS,
NON-ROUTINE INTERVENTIONS AND EVENTS (E.G. MAINTENANCE, ELECTRIC SHUT
DOWN, PERSONAL INTRUSION ETC)
• NO OF PERSONNEL & THEIR ACTIVITIES, SHIFT CHANGES, BREAKS, GOWN
CHANGES
• REPRESENTATIVE NUMBER OF ASEPTIC ADDITIONS (E.G. CHARGING CONTAINERS,
CLOSURES, STERILE INGREDIENTS) OR TRANSFERS
• ASEPTIC EQUIPMENT CONNECTIONS/DISCONNECTIONS
• ASEPTIC SAMPLE COLLECTIONS
• LINE SPEED AND CONFIGURATION
• WEIGHT CHECKS
• CONTAINER CLOSURE SYSTEMS
• WRITTEN BATCH RECORD DOCUMENTING CONDITIONS AND ACTIVITIES
27
ASEPTIC PROCESSING

ASEPTIC PROCESSING
DURATION
• DEPENDS ON TYPE OF OPERATION
• PROCESSES - SUFFICIENT TIME TO INCLUDE MANIPULATIONS AND INTERVENTIONS
• FOR CONVENTIONAL OPERATIONS SHOULD INCLUDE THE TOTAL FILLING TIME
SIZE
• 5000 - 10000 GENERALLY ACCEPTABLE OR BATCH SIZE IF <5000
• FOR MANUALLY INTENSIVE PROCESSES LARGER NUMBERS SHOULD BE FILLED
• LOWER NUMBERS CAN BE FILLED FOR ISOLATORS
28
PROCESS VALIDATION (MEDIA FILL TEST )
FREQUENCY AND NUMBER
– THREE INITIAL, CONSECUTIVE PER SHIFT
– SUBSEQUENTLY SEMI-ANNUAL PER SHIFT AND PROCESS
– ALL PERSONNEL SHOULD PARTICIPATE AT LEAST ANNUALLY, CONSISTENT WITH ROUTINE
DUTIES
– CHANGES SHOULD BE ASSESSED AND REVALIDATION CARRIED OUT AS REQUIRED
LINE SPEED
– SPEED DEPENDS ON TYPE OF PROCESS

ASEPTIC PROCESSING
• ENVIRONMENTAL CONDITIONS
• REPRESENTATIVE OF ACTUAL PRODUCTION CONDITIONS (NO. OF
PERSONNEL, ACTIVITY LEVELS ETC) - NO SPECIAL PRECAUTIONS (NOT
INCLUDING ADJUSTMENT OF HVAC)
• IF NITROGEN USED FOR OVERLAYING/PURGING NEED TO SUBSTITUTE WITH
AIR
• MEDIA
• ANAEROBIC MEDIA SHOULD BE CONSIDERED UNDER CERTAIN
CIRCUMSTANCES
• SHOULD BE TESTED FOR GROWTH PROMOTING PROPERTIES (INCLUDING
FACTORY ISOLATES)

ASEPTIC PROCESSING
• INCUBATION, EXAMINATION
• IN THE RANGE 20-35ºC (20-25ºC FOR FUNGAL & 30-35ºC FOR BACTERIA.
• IF TWO TEMPERATURES ARE USED, LOWER TEMPERATURE FIRST
• INSPECTION BY QUALIFIED PERSONNEL.
• ALL INTEGRAL UNITS SHOULD BE INCUBATED. SHOULD BE JUSTIFICATION
FOR ANY UNITS NOT INCUBATED.
• UNITS REMOVED (AND NOT INCUBATED) SHOULD BE CONSISTENT WITH
ROUTINE PRACTICES (ALTHOUGH INCUBATION WOULD GIVE
INFORMATION REGARDING RISK OF INTERVENTION)
• BATCH RECONCILIATION

ASEPTIC PROCESSING
• INTERPRETATION OF RESULTS
• WHEN FILLING FEWER THAN 5000 UNITS:
• NO CONTAMINATED UNITS SHOULD BE DETECTED
• ONE (1) CONTAMINATED UNIT IS CONSIDERED CAUSE
FOR REVALIDATION, FOLLOWING AN INVESTIGATION
• WHEN FILLING FROM 5000-10000 UNITS
• ONE (1) CONTAMINATED UNIT SHOULD RESULT IN AN INVESTIGATION, INCLUDING
CONSIDERATION OF A REPEAT MEDIA FILL
• TWO (2) CONTAMINATED UNITS ARE CONSIDERED CAUSE FOR REVALIDATION,
FOLLOWING INVESTIGATION
• WHEN FILLING MORE THAN 10000 UNITS
• ONE (1) CONTAMINATED UNIT SHOULD RESULT IN AN INVESTIGATION
• TWO (2) CONTAMINATED UNITS ARE CONSIDERED CAUSE FOR REVALIDATION,
FOLLOWING INVESTIGATION

ASEPTIC PROCESSING
• INTERPRETATION OF RESULTS
• MEDIA FILLS SHOULD BE OBSERVED BY QC AND CONTAMINATED UNITS
RECONCILABLE WITH TIME AND ACTIVITY BEING SIMULATED (VIDEO MAY
HELP)
• IDEALLY - NO CONTAMINATION. ANY CONTAMINATION SHOULD BE
INVESTIGATED.
• ANY ORGANISMS ISOLATED SHOULD BE IDENTIFIED TO SPECIES LEVEL
(GENOTYPIC IDENTIFICATION)
• INVALIDATION OF A MEDIA FILL RUN SHOULD BE RARE

33
CASE STUDY 01

Aseptic Processing & Media Fill

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