Sterile Pharmaceutical
Products
EU GMP guide, Annex 1
25 November 2008
Dr. Ursula Koller 1
Objectives
To review basic GMP requirements in the manufacture
of sterile pharmaceutical products
To review air classifications for activities related to the
manufacture of sterile products
To review the different types of sterilization methods
To review quality assurance aspects in the
manufacture and control of sterile products
To consider current issues applicable in your country
Dr. Ursula Koller 2
GMP Requirements for Sterile Products
Additional rather than replacement
Specific points relating to minimizing risks of
contamination
microbiological
particulate matter
Pyrogen
Skills, training and attitudes of personnel involved are
crucial
Dr. Ursula Koller 3
General Considerations - 1
Production in clean areas
Appropriate standard of cleanliness
Filtered air supplied
Airlocks for entry
personnel and/or equipment
materials
Separate areas for operations
component preparation (containers and closures)
product preparation
filling, sterilization, etc.
Terminally sterilised products
Aseptically production at some or all stages
Dr. Ursula Koller 4
General Considerations - 2
To meet “in-operation” conditions, areas designed to reach
certain specified air-cleanliness levels in “at-rest” stage
“At-rest” state is without operating personnel
“in-operation” state with the specified number of personnel
working
4 grades for manufacturing sterile medicinal products:
Grade A:
High risk operations, e.g. filling zone, stopper bowls, open
containers (vials, ampoules, bottles), making aseptic
connections
Laminar air flow work station with homogeneous air speed of
0.36 to 0.54 m/s at the working position in open clean room
applications
Maintenance of laminarity demonstrated and validated
In glove boxes or closed isolators unidirectional flow and lower
velocities may be used
Dr. Ursula Koller 5
General Considerations - 3
Grade B:
Background environment for grade A zones for aseptic
preparation and filling
Grade C:
Clean areas for carrying out less critical stages in the
manufacturing of sterile products such as
Preparation of solutions to be filtered (aseptic operations; see
paragraph 31-35)
Preparation of solutions when unusually at risk; Filling of products
(terminally sterilised products; see paragraph 28-30)
Grade D:
Clean areas for carrying out less critical stages in the
manufacturing of sterile products such as
Handling of components after washing (aseptic operations)
Preparation of solutions and components for subsequent filling
(terminally sterilised products)
Dr. Ursula Koller 6
Clean room and clean air device
CLASSIFICATION -1
Clean rooms and clean air devices classified according to EN
ISO 14644-1
Classification is differentiated from operational process
environmental monitoring
Maximum permitted number of airborne particles per m3
equal to or greater than the tabulated size:
Dr. Ursula Koller 7
Airborne particle classification - 2
Dr. Ursula Koller 8
Clean room and clean air device
CLASSIFICATION -3
Classification in grade A zones
Minimum sample volume of 1 m3 should be taken per sample
location
For grade A the airborne particle classification is ISO 4.8
dictated by the limit for particles ≥ 5.0 µm
Grade B (at rest)
ISO 5 for both considered particle sizes
Grade C (at rest and in operation)
Airborne particle classification is ISO 7 and ISO 8 respectively
Grade D (at rest)
Airborne particle classification is ISO 8
For classification purposes EN ISO 146444-1 methodology
defines both the minimum number of sample locations and
the sample size based on the class limit of the larger
considered particle size and the method of evaluation of the
data collected. Dr. Ursula Koller 9
Clean room and clean air device
CLASSIFICATION -4
Portable particle counters with a short length of sample
tubing used for classification purposes
Isokinetic sample heads are preferred in unidirectional
airflow systems
“In operation” may be demonstrated during normal
operations, simulated operations or during media fill as
worst case simulation is required for this
EN ISO 14644-2 provides information on testing to
demonstrate continued compliance with the assigned
cleanliness classifications
Dr. Ursula Koller 10
Clean room and clean air device monitoring
–1
Monitoring of clean areas routinely in operation
Monitoring locations defined based on a risk assessment
and the results obtained during area classification
Monitoring of personnel and surfaces after critical
operations
Results considered when batch release is done
Additional microbiological monitoring required outside
production operations, e.g. after validation of systems,
cleaning, sanitisation
Dr. Ursula Koller 11
Clean room and clean air device
monitoring – 2
Frequent monitoring in areas where aseptic operations
are carried out
settle plates, volumetric air samples, surface
sampling (swabs and contact plates)
sampling methods should not contaminate the
area
Dr. Ursula Koller 12
Clean room and clean air device
monitoring – 3
Grade A zones
Particle monitoring undertaken for full duration of critical
processing, including equipment assembling
Monitoring during simulated operations (media fill)
Monitoring frequency and sample size so that all interventions,
transient events and any system deterioration are captured
Demonstration of low limits of particles ≥ 5.0 µm at filling point not
always possible due to particles or droplets from product itself
Grade B zones
Similar system of monitoring but determined by effectiveness of
segregation between the adjacent grade A and B zones
Dr. Ursula Koller 13
Clean room and clean air device
monitoring – 4
Grade C and D zones
Monitoring in operation performed according to a risk assessment
Requirements and action/alert limits depend on nation of
operations carried out, a “clean up period” is recommended
“clean up period”
Particle limits given in table 1 for the “at rest” state achieved after
short “clean up period” of 15 – 20 min in a unmanned state after
operations are completed
Airborne particle monitoring system, sample size,
particle concentration is described in Annex 1 in detail
(11, 12, 13)
Dr. Ursula Koller 14
Environmental Monitoring – 1
Physical
Particulate matter
Differential pressures
Air changes, airflow patterns
Clean up time/recovery
Filter integrity
Temperature and relative humidity depending on
product and nature of operations
Airflow velocity
Dr. Ursula Koller 15
Environmental Monitoring – 2
Appropriate alert and action limits set for results of
particulate and microbiological monitoring
Limits exceeded procedures describing the corrective
action to be taken
Dr. Ursula Koller 16
Limits for microbiological monitoring of
clean areas in operation
Table 1. Recommended limits for microbial contamination (a)
(a) Average values
(b) Individual settle plates may be exposed for less than 4 hours
Aseptic preparation - 1
Air samples
Surface swabs
Personnel swabs
Dr. Ursula Koller 18
Personnel - 1
Minimum number of personnel in clean areas (verifying
during media fill)
especially during aseptic processing
Inspections and controls from outside
Training to all including cleaning and maintenance staff
initial and regular
manufacturing, hygiene, basics of microbiology
Special cases
Instruction and supervision in case of outside
staff
Dr. Ursula Koller 19
Personnel - 2
High standards of hygiene and cleanliness
Report of any condition may causing shedding of of
abnormal numbers or types of contaminants to supervisor
Periodic health checks for such conditions
No introduction of microbiological hazards
No outdoor clothing
No wristwatches, jewellery and make-up in clean areas
Written procedure for changing and washing
Dr. Ursula Koller 20
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
– no fibres to be shed
Dr. Ursula Koller 21
Personnel - 4
Clothing of appropriate quality cont:
Grade A and B
Headgear (totally enclose hair, tucked into the neck of
suit), beard and moustache covered
face masks, (goggles)
Sterilised , non-powdered gloves
Sterilised/disinfected footwear
Trouser-legs tucked into the footwear
Garment sleeves tucked into the gloves
Arm cuffs/sleeves
not shedding fibres, and retain particles shed by
operators
Dr. Ursula Koller 22
Personnel - 5
No outdoor clothing not in change rooms
Change of clean, sterile protective garments at every
working session (breaks), or once a day (if supportive
data available)
Change gloves and masks at every working session
Regular disinfection of gloves during operations (e.g.
after each intervention into class A, whenever using
scissors, tweezers, etc.
Washing and sterilising of garments – separate laundry
facility – written, validated procedures (no damage of
fibres)
Dr. Ursula Koller 23
Premises - 1
Design
avoid unnecessary entry of supervisors and control
personnel
operations observed from outside
In clean areas, all exposed surfaces
smooth, impervious, unbroken
minimize shedding and accumulation of particles,
microorganisms
permit cleaning and disinfection
no uncleanable recesses, ledges, shelves, cupboards,
equipment only required equipment
sliding doors undesirable
false ceilings sealed
Dr. Ursula Koller 24
Premises - 2
In clean areas, all exposed surfaces
proper installation of pipes and ducts, no recesses, no
unsealed openings
sinks and drains are prohibited in Grade A and B areas
in other areas where installed:
– correct design, location, maintenance
– air breaks between machine or sink and the drains
– effective cleanable traps or water seals to prevent
backflow
– floor channels open and easily cleanable
Dr. Ursula Koller 25
Premises - 3
Changing rooms
designed as airlocks
Provide physical separation of different stages of
changing (unclassified to grade B) to minimise
microbial and particulate contamination of
protective clothing
effective flushing with filtered air
Final stage of changing room should at-rest state,
be the same grade as the area leading into (class B
changing room leads into room class B)
separate rooms for entry and exit sometimes
desirable
Dr. Ursula Koller 26
Premises - 4
Changing rooms
hand washing facilities provided in first stage of
changing rooms
interlocking system for doors
visual and/or audible warning system
Use filtered air supply to maintain positive pressure
cascade
Air flow relative to surrounding areas of a lower grade
under all operational conditions (higher pressure inside
the cleaner room)
Effective flushing of the area
Dr. Ursula Koller 27
Premises – 5
Pressure differential approximately 10 to 15 Pa
Zone of greatest risk is the immediate environment to
which a product or cleaned components which contact
the product are exposed
Pressure cascade may be different for pathogenic,
highly toxic, radioactive materials, live viral or bacterial
materials
Decontamination procedures – air, equipment,
garments
Dr. Ursula Koller 28
Premises – 6
Qualification of HVAC system including airflow patterns
To demonstrate that there is no risk of
contamination to the product
Warning system to indicate failure in air supply
Pressure indicators – results regularly recorded
Restricted access – e.g. use of barriers
Dr. Ursula Koller 29
Equipment - 1
No pass of conveyer belts between grads A and B
and a processing area of lower grade (unless it is
continually sterilised before moving into clean area)
Effective sterilization of equipment
Maintenance and repairs from outside the clean
area
if taken apart, sterilising after reassembling
Maintenance within the clean area area cleaned,
disinfected and/or sterilised prior to processing
Always use clean instruments and tools
Dr. Ursula Koller 30
Equipment – 2
All equipment, air handling and filtration
systems, sterilizers, air vent and gas filters
water treatment systems (incl. generation,
storage and distribution loop) need planned
maintenance, validation and monitoring
Dr. Ursula Koller 31
Equipment – 3
Water treatment plants and distribution system
Appropriate design, construction,
maintenance
operation within the designed capacity
testing programme
Water for Injection (WFI)
produced, stored, distributed – prevention of
growth of microorganisms
constant circulation at temperature above
70°C
Dr. Ursula Koller 32
Sanitation
Frequent, thorough cleaning of areas necessary
Written programme
Using more than one type of disinfectant regular
monitoring of disinfectants to detect resistant strains of
microorganisms
Chemical disinfection
Monitoring of disinfectants and detergents
Dilutions of disinfectants and detergents
clean containers, stored for defined periods of time
Sterilized before use, when used in Grade A or B areas
Fumigation may be useful to reduce microbiological
contamination in inaccessible places
Dr. Ursula Koller 33
Group session 1
You are asked to visit a factory producing the following
product lines:
sterile eye drops
Describe the type of facility you would expect to find
List the typical rooms, their purpose and air
classification
Dr. Ursula Koller 34
Possible Issues (Group Session) 1
Poor design of the building
Poor design of the systems, e.g. water, HVAC
Flow of personnel
Flow of material
No validation or qualification
Old facilities not complying with current requirements
Dr. Ursula Koller 35
Possible Issues (Group Session) 2
Particulate levels/microorganisms
Differential pressures
Air changes
Temperature/humidity
Dr. Ursula Koller 36
Two categories of manufacturing
operations
Terminally sterilized
prepared, filled and sterilized
Aseptic preparation
some or all stages
Dr. Ursula Koller 37
Manufacture of sterile preparations
To reach Grade B, C and D, the number of air changes
should be appropriate to the size of the area, number of
personnel, equipment present
Minimum of 20 air changes per hour
Clean-up time about 15 – 20 minutes
Good airflow pattern in the area
HEPA filtered air
Suitable methods to determine particulate matter and
micro
e.g. EU, ISO, Japan, USA
Dr. Ursula Koller 38
Manufacture of sterile preparations
Control particulate during operation
Monitoring during operation
Alert and action limits for particulate and micro
Action taken when exceeded
Area grades should be proven (e.g. validation runs,
media fills, environment, time limits - based on
microbiological contamination/bioburden found)
Dr. Ursula Koller 39
Processing - 1
Minimize contamination - all stages including before
sterilization and during processing
Minimize activities
staff movement controlled and methodical
avoid shedding of particles
Temperature and humidity comfortable
Starting materials – microbiological contamination
should be minimal
Containers and materials liable to generate fibres
minimised in the clean areas
Dr. Ursula Koller 40
Processing - 2
Validation – should not compromise the processes
Aseptic process validation: sterile media fill (“broth
fills”)
simulate actual operation – intimate as closely as
possible
simulate worst expected condition e.g. interventions
use appropriate medium/media
Process simulation tests as initial validation with 3
consecutive satisfactory simulation tests per shift
revalidation: periodic and after change of HVAC,
equipment, process and number of shifts
Normally twice per year per shift and process
Dr. Ursula Koller 41
Processing - 3
Media fill
sufficient number of units
equal to batch size (small batches)
Target: zero growth!!
Filling fewer than 5.000 units: no contamination
Filling 5.000 to 10.000 units:
One contaminated unit Investigation, incl. consideration of a
repeat media fill
Two contaminated units repeat media fill (revalidation) and
investigation
Filling more than 10.000 units:
One contaminated unit investigation
Two contaminated repeat media fill (revalidation) and
investigation
Dr. Ursula Koller 42
Processing - 4
Media fill cont:
Investigation of media fill failures should include
potential impact on sterility assurance of all
batches manufactured since the last successful
media fill
Intermittent incidents of microbial
contamination may be indicative of low-level
contamination which is to be investigated
Dr. Ursula Koller 43
Processing - 5
Water sources, water treatment systems and treated
water
Monitored regularly
chemicals
biological contamination
endotoxins
Water specification
Records of results and action taken
Dr. Ursula Koller 44
Processing - 6
Cleaning of components, bulk product containers and
equipment
no recontamination after final cleaning
stage properly identified (due date for
cleaning/sanitising)
sterilized when used in aseptic areas
Components, containers, equipment used in clean areas
(aseptic work) sterilised, passed through double-ended
sterilizers sealed into the wall (transfer hatch), or use
triple wrapping
Gas used to purge solution or blanket a product – passed
through a sterilizing filter
Dr. Ursula Koller 45
Processing - 7
Bioburden monitored
products: before sterilization
working limits established
solutions to be filtered before filling (especially LVP,
aseptic preparations)
pressure release outlets – hydrophobic
microbiological air filters
Monitored as per specification
Time between preparation of solution and filtration minimised
Set maximum permissible time for each product
Dr. Ursula Koller 46
Processing - 8
Time intervals: components, bulk containers,
equipment
Washing and drying and sterilization; and
sterilization and use
as short as possible
time limit validated
Time intervals: product
Start of preparation of solution and sterilization
(filtration)
as short as possible
maximum time set for each product
Dr. Ursula Koller 47
Sterilization - 1
Methods of sterilization
moist or dry heat
irradiation (ionizing radiation)
sterilizing gaseous agents (e.g. ethylene oxide)
filtration with subsequent aseptic filling
Whenever possible: terminal sterilization by heat in
their final container - method of choice
Dr. Ursula Koller 48
Sterilization - 2
Validation
all sterilization processes
special attention when non-pharmacopoeial methods
are used
non-aqueous or oily solutions
Before the method is adopted – its suitability and
efficacy demonstrated with desired conditions
all parts of the load
each type of load
physical measurements and biological indicators
(where appropriate)
verified at least annually and after change
records maintained
Dr. Ursula Koller 49
Sterilization - 3
For effective sterilization
Whole of the material subjected to the treatment
Biological indicators
Additional method of monitoring
Storage and use, quality checked through positive
control
Risk of contamination
Dr. Ursula Koller 50
Sterilization - 4
Differentiation between sterilized and not-yet-sterilized
products
Each basket/tray or other carrier, properly labelled
name of material
batch number
sterilization status
Use of autoclave tape
Sterilization records for each run – approved as part of the
batch release procedure
Dr. Ursula Koller 51
Aseptic processing and sterilization by
filtration
Aseptic processing
Objective is to maintain the sterility of a product,
assembled from sterile components
Operating conditions so as to prevent microbial
contamination
What do you think are the aspects that require careful
attention?
Dr. Ursula Koller 52
Aseptic processing and sterilization by
filtration
Aseptic processing (2)
Careful attention to:
Environment
Personnel
Critical surfaces
Container/closure sterilization
Transfer procedures
Maximum holding period before filling
Dr. Ursula Koller 53
Aseptic preparation (Annex 1, 31 -35)
Aseptic preparation
Grade Preparation Remark
A Sterile starting materials and (Unless subjected to
(in B components sterilization or filtration
background) through a microorganism
retaining filter later in the
process)
A Preparation of solutions (if
(in B not to be sterile filtered later)
background)
A Handling and filling of
(in B aseptically prepared
background) products,
A Handling of exposed sterile
(in B equipment
background)
A Transfer of partially closed E.g. in freeze drying
(in B containers, before complete (Grade B environment if
background) stoppering in sealed transfer trays)
Sterilization by Filtration - 1
Through a sterile filter of 0,22 µm or less, into
previously sterilized containers
remove bacteria and moulds
not all viruses or mycoplasmas
Consider complementing with some degree of heat
treatment
Double filter layer or second filtration advisable, just
directly before filling - no fibre shedding filters
Filter integrity testing before use and confirmed
immediately after use by an appropriate method
(bubble point, diffusive flow, pressure hold test)
Dr. Ursula Koller 56
Sterilization by Filtration - 2
Validation to include
time taken to filter a known volume
pressure difference to be used across the filter
Significant differences to be noted and investigated,
recorded in batch records
Integrity of gas and air vent filters checked after use,
other filters at appropriate intervals
Dr. Ursula Koller 57
Sterilization by Filtration - 3
Same filter not used for more than one working day,
unless validated
No filter interaction with product, e.g.
removal of ingredients
releasing substances into product
Dr. Ursula Koller 58
Group session 2
Considering the same factory as in the previous group
session, discuss the process of sterilization by filtration
List all the sterile items needed for this process (and
indicate the choice of sterilization process)
What are the key features you should find in each
sterilization situation?
Discuss the relevance, need, and the extent of
qualification and validation required
Dr. Ursula Koller 59
Possible Issues (Group session)
Autoclave - no pressure gauge
Autoclave - no temperature recorder
Autoclave - superheated steam
Clean room - pressure differentials
Exposure for settle plates
Interlocks turned off
Rusty Laminar airflow cabinets
HEPA filters not checked regularly
Dr. Ursula Koller 60
Finishing of products
Containers closed by means of validated methods
Samples checked for integrity
Maintenance of vacuum (where applicable) checked
Parenteral products inspected individually
Visual inspection under suitable and controlled conditions
illumination and background
eyesight checks of operators
allowed frequent breaks
Other methods
validated, and equipment performance checked at
intervals
results recorded
Dr. Ursula Koller 61
Quality Control 1
Samples for sterility testing should be representative
From parts of the batch, most at risk
aseptic filling - at beginning and end of batch filling,
and after interruptions
heat sterilized – coolest part of the load
Sterility of the batch ensured through validation
validated sterilization cycle
media fill
Sterility test procedure as per pharmacopoeia, and
validated for each product
Batch processing records, sterility testing records,
environmental records should be reviewed
Dr. Ursula Koller 62
Quality Control 2
Endotoxin testing for injectable products
water for injection, intermediate and finished
product
Always for large volume infusion solutions
Pharmacopoeia method, validated for each product
Failure of the test – investigation
Corrective action
Dr. Ursula Koller 63
Group session 3
Considering the same factory as in the previous group
sessions, devise a plan for monitoring of the facility
List the parameters to be tested, tests to be used,
acceptance criteria and frequency of testing
Dr. Ursula Koller 64
