Aseptic Processing

Mrs Robyn Isaacson

1 Manufacture of sterile medicines –

 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

2 Manufacture of sterile medicines –

 Aseptic Processing - Overview

Aseptic processing
• Objective is to maintain the sterility of a product,
assembled from sterile components
• Operating conditions so as to prevent microbial
contamination

3 Manufacture of sterile medicines –

 Aseptic Processing - Overview
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
– Specific issues relating to Isolators, BFS and Bulk
4 Manufacture of sterile medicines –
 Manufacturing Environment
Classification of Clean Areas
– Comparison of classifications

WHO GMP US 209E US Customary ISO/TC (209) EEC GMP

ISO 14644
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

5 Manufacture of sterile medicines –

 Manufacturing Environment
Classification of Clean Areas
– Classified in terms of airborne particles (Table 2)
Grade At rest In operation

maximum permitted number of particles/m 3

0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm >5µ
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

6 Manufacture of sterile medicines –

 Manufacturing Environment

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.

7 Manufacture of sterile medicines –

 Manufacturing Environment
• 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)

8 Manufacture of sterile medicines –

 Manufacturing Environment
• Limits for viable particles (microbiological
contamination)
Grade Air sample Settle plates (90mm Contact plates Glove print
(CFU/m3) diameter) (55mm (5 fingers)
(CFU/4hours) diameter) (CFU/glove)
(CFU/plate)
A <3 <3 <3 <3
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 less than 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
9 Manufacture of sterile medicines –
 Manufacturing Environment
Environmental Monitoring
• Physical
– Particulate matter
– Differential pressures
– Air changes, airflow patterns
– Clean up time/recovery
– Temperature and relative humidity
– Airflow velocity

10 Manufacture of sterile medicines –

 Manufacturing Environment
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

11 Manufacture of sterile medicines –

 Manufacturing Environment
Environmental Monitoring - Physical
• 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

12 Manufacture of sterile medicines –

 Manufacturing Environment
Environmental Monitoring - Physical
• 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

13 Manufacture of sterile medicines –

 Manufacturing Environment
Environmental Monitoring - Physical
• 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)

14 Manufacture of sterile medicines –

 Manufacturing Environment
Personnel
• Minimum number of personnel in clean areas
– especially during aseptic processing
• Inspections and controls from outside
• Training to all including cleaning and
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)
15 Manufacture of sterile medicines –
 Manufacturing Environment
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
16 Manufacture of sterile medicines –
 Manufacturing Environment
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

17 Manufacture of sterile medicines –

 Manufacturing Environment

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

18 Manufacture of sterile medicines –

 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

19 Manufacture of sterile medicines –

 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
– do not remove all viruses or mycoplasmas
• filtration should be carried out under positive
pressure

20 Manufacture of sterile medicines –

 Aseptic Processing
Preparation and Filtration of Solutions (2)
• consideration should be given to complementing
filtration process with some form of heat treatment
• Double filter or second filter at point of fill advisable
• Fitlers should not shed particles, asbestos containing
filters should not be used
• Same filter should not be used for more than one day
unless validated
• If bulk product is stored in sealed vessels, pressure
release outlets should have hydrophobic microbial
retentive air filters

21 Manufacture of sterile medicines –

 Aseptic Processing
Preparation and Filtration of Solutions (3)
• 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)

22 Manufacture of sterile medicines –

 Aseptic Processing
Preparation and Filtration of Solutions (4)
• Filling of solution may be followed by lyophilization
(freeze drying)
– stoppers partially seated, product transferred to
lyophilizer (Grade A/B conditions)
– Release of air/nitrogen into lyophilizer chamber at
completion of process should be through sterilizing
filter

23 Manufacture of sterile medicines –

 Aseptic Processing
Prefiltration Bioburden (natural microbial load)
• Limits should be stated and testing should be carried
out on each batch
• Frequency may be reduced after satisfactory history
is established
– and biobuden testing performed on components
• Should include action and alert limits (usually differ
by a factor of 10) and action taken if limits are
exceeded
• Limits should reasonably reflect bioburden routinely
achieved

24 Manufacture of sterile medicines –

 Aseptic Processing

Prefiltation Bioburden (2)

• No defined “maximum” limit but the limit should not
exceed the validated retention capability of the filter
• Bioburden controls should also be included in “in-
process” controls
– particularly when product supports microbial growth
and/or manufacturing process involves use of culture
media
• Excessive bioburden can have adverse effect on the
quality of the product and cause excessive levels of
endotoxins/pyrogens

25 Manufacture of sterile medicines –

 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
lyphilization chamber
• filter intergrity shoud be verified before filtration and
confirmed after filtration
– bubble point
– pressure hold
– forward flow
• methods are defined by filter manufacturers and limits
determined during filter validation

26 Manufacture of sterile medicines –

 Aseptic Processing
Filter Validaton
• Filter must be validated to demonstrate ability to
remove bacteria
– most common method is to show that filter can retain a
microbiological challenge of 107 CFU of Brevundimonas
diminuta per cm2 of the filter surface
– a bioburden isolate may be more appropriate for filter
retention studies than Brevundimonas diminuta
– Challenge concentration is intended to provide a margin
of safety well beyond what would be expected in
production
– preferably the microbial challenge is added to the fully
formulated product which is then passed through the
filter

27 Manufacture of sterile medicines –

 Aseptic Processing
Filter validation (2)
– if the product is bactericidal, product should be passed
through the filter first followed by modified product
containing the microbial challenge (after removing any
bactericidal activity remaining on the filter)
– filter validation should be carried out under worst case
conditions e.g. maximum allowed filtration time and
maximum pressure
– integrity testing specification for routine filtration
should correlate with that identified during filter
validation

28 Manufacture of sterile medicines –

 Aseptic Processing
Equipment/container preparation and
sterilization
• All equipment (including lyophilizers) and product
containers/closures should be sterilized using
validated cycles
– same requirements apply for equipment sterilization that
apply to terminally sterilized product
– 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

29 Manufacture of sterile medicines –

 Aseptic Processing

Equipment/container preparation and

sterilization (2)
• CIP/SIP processes
– particular attention to deadlegs - different orientation
requirements for CIP and SIP
• heat tunnels often used for
sterilization/depyrogenation of glass vials/bottles
– usually high temperature for short period of time
– need to consider speed of conveyor
– validation of depyrogenation (3 logs endotoxin units)
• worst case locations
– tunnel supplied with HEPA filtered air

30 Manufacture of sterile medicines –

 Aseptic Processing
Equipment/container preparation and
sterilization (2)
• 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)

31 Manufacture of sterile medicines –

 Aseptic Processing
Process Validation
• 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
– modifications must be made for different dosage
formats e.g. lyophilized products, ointments, sterile
bulks, eye drops filled into semi-transparent/opaque
containers, biological products

32 Manufacture of sterile medicines –

 Aseptic Processing
Process Validation (2)
• 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, stoppages, etc)
– Lyophilisation
– Aseptic equipment assembly

33 Manufacture of sterile medicines –

 Aseptic Processing
Process Validation (3)
• Worst case activities (cont)
– No of personnel and 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

34 Manufacture of sterile medicines –

 Aseptic Processing

Process Validation (4)

• Worst case activities (cont)
– Weight checks
– Container closure systems
– Specific provisions in processing instructions
• Written batch record documenting conditions and
activities
• Should not be used to justify risky practices

35 Manufacture of sterile medicines –

 Aseptic Processing

Process Validation (5)

Duration
– Depends on type of operation
– BFS, Isolator 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

36 Manufacture of sterile medicines –

 Aseptic Processing
Process Validation (6)
• 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

37 Manufacture of sterile medicines –

 Aseptic Processing

Process Validation (7)

• 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)

38 Manufacture of sterile medicines –

 Aseptic Processing
Process Validation (8)
• Incubation, Examination
– In the range 20-35ºC.
– 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

39 Manufacture of sterile medicines –

 Aseptic Processing
Process Validation (9)
• 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

40 Manufacture of sterile medicines –

 Aseptic Processing

Process Validation (10)

• 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

41 Manufacture of sterile medicines –

 Aseptic Processing

Process Validation (11)

• Batch Record Review
– Process and environmental control activities
should be included in batch records and reviewed
as part of batch release
• In-process and laboratory control results
• Environmental and personnel monitoring data
• Output from support systems(HEPA/HVAC, WFI, steam
generator)
• Equipment function (batch alarm reports, filter integrity)
• Interventions, Deviations, Stoppages - duration and
associated time
• Written instructions regarding need for line clearances
• Disruptions to power supply

42 Manufacture of sterile medicines –

 Aseptic Processing
Additional issues specific to Isolator and
BFS Technologies
• Isolators
– Decontamination process requires a 4-6 log
reduction of appropriate Biological Indicator (BI)
– Minimum 6 log reduction of BI if surface is to be
free of viable organisms
– Significant focus on glove integrity - daily checks,
second pair of gloves inside isolator glove
– Traditional aseptic vigilance should be maintained

43 Manufacture of sterile medicines –

 Aseptic Processing
• Blow-Fill-Seal (BFS)
– Located in a Grade D environment
– Critial zone should meet Grade A (microbiological)
requirements (particle count requirements may be
difficult to meet in operation)
– Operators meet Grade C garment requirements
– Validation of extrusion process should
demonstrate destruction of endotoxin and spore
challenges in the polymeric material
– Final inspection should be capable of detecting
leakers

44 Manufacture of sterile medicines –

 Aseptic Processing
• Issues relating to Aseptic Bulk Processing
• Applies to products which can not be filtered at point of
fill and require aseptic processing throughout entire
manufacturing process.
• Entire aseptic process should be subject to process
simulation studies under worst case conditions
(maximum duration of "open" operations, maximum no
of operators)
• Process simulations should incorporate storage and
transport of bulk.
• Multiple uses of the same bulk with storage in between
should also be included in process simulations
• Assurance of bulk vessel integrity for specified holding
times.

45 Manufacture of sterile medicines –

 Aseptic Processing
• Bulk Processing (2)
• Process simulation for formulation stage should be
performed at least twice per year.
– Cellular therapies, cell derived products etc
• products released before results of sterility tests
known (also TPNs, radioactive preps, cytotoxics)
• should be manufactured in a closed system
• Additional testing
– sterility testing of intermediates
– microscopic examination (e.g. gram stain)
– endotoxin testing

46 Manufacture of sterile medicines –

 Useful Publications
• PIC/S Recommendation on the Validation of Aseptic
Processes
• FDA Guidance for Industry- Sterile Drug Products Produced
by Aseptic Processing - Current Good Manufacturing
Process
• ISO 13408 Aseptic Processing of Health Care Products
– Part 1: General Requirements
– Part 2: Filtration
– Part 3: Lyophilization
– Part 4: Clean-In-Place Technologies
– Part 5: Sterilization-In-Place
– Part 6: Isolator Systems

47 Manufacture of sterile medicines –