Aseptic Processing Guide

Aseptic Processing
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
Manufacture of sterile medicines Advanced workshop for SFDA GMP

inspectors - Nanjing, November 2009

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
Specific issues relating to Isolators, BFS and Bulk

Manufacturing Environment
Classification of Clean Areas
Comparison of classifications
Table 1
5

Classification of Clean Areas
Classified in terms of airborne particles (Table 2)
At rest - production equipment installed and operating

In operation - Installed equipment functioning in defined
operating mode and specified number of personnel present
6

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)
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

Environmental Monitoring
Physical
Particulate matter
Differential pressures
Air changes, airflow patterns
Clean up time/recovery
Temperature and relative humidity
Airflow velocity
10

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

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

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

Temperature and Relative Humidity
Ambient temperature and humidity should not be
uncomfortably high (could cause operators to
generate particles) (18C)
Airflow velocity
Laminar airflow workstation air speed of approx
0.45m/s 20% at working position (guidance value)
14

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

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

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

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

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

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 0.22m (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

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

Aseptic Processing
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

Aseptic Processing
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

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

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 inprocess 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

Aseptic Processing
Filter integrity
Filters of 0.22m 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

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

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

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

Aseptic Processing
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

Aseptic Processing
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
31
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
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

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

Aseptic Processing
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

Aseptic Processing
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

Aseptic Processing
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

Aseptic Processing
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

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)
38

Aseptic Processing
Incubation, Examination
In the range 20-35C.
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

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
40

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
41

Aseptic Processing
Batch Record Review
Process and environmental control activities
should be included in batch records and reviewed
as part of batch release
42
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

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

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

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

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

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
47
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


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Aseptic Processing

Aseptic Processing - Overview

Two categories of sterile products

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Aseptic Processing - Overview

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Aseptic Processing - Overview

Preparation and filtration of solutions

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

At rest - production equipment installed and operating

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Grade C (equivalent to Class 10,000, ISO 7):

Grade B (equivalent to Class 100, ISO 5):

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Each grade of cleanroom has specifications for

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Inspections and controls from outside

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Periodic health checks

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Regular microbiological monitoring of operators

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

filtration should be carried out under positive pressure

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Should include action and alert limits (usually differ

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Excessive bioburden can have adverse effect on the

Manufacture of sterile medicines Advanced workshop for SFDA GMP

filter intergrity shoud be verified before filtration and

methods are defined by filter manufacturers and limits

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

heat tunnels often used for

tunnel supplied with HEPA filtered air

Manufacture of sterile medicines Advanced workshop for SFDA GMP

equipment should be designed to be easily assembled and

rinse water should be WFI grade

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Written batch record documenting conditions and

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

Manufacture of sterile medicines Advanced workshop for SFDA GMP

When filling from 5000-10000 units

When filling more than 10000 units

Manufacture of sterile medicines Advanced workshop for SFDA GMP