Sterile Pharmaceutical Products

Supplementary Training Modules on
Good Manufacturing Practice
Sterile Pharmaceutical Products
Annex 6. TRS 902, 2002
Sterile | Slide 1 of 62 January 2006

Sterile Production
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

Sterile Production
GMP Requirements for Sterile Products

 Additional rather than replacement
 Specific points relating to minimizing risks of

contamination
– microbiological
– particulate matter
– pyrogen

Sterile Production
General Considerations
 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. 1.1 – 1-2

Sterile Production
Premises
 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
– Sliding doors undesirable
– False ceilings sealed 9.1 – 9.6

Sterile Production
Premises
 In clean areas, all exposed surfaces (2):
– Proper installation of pipes and ducts, no recesses, no
unsealed openings
– Sinks and drains avoided, and excluded in Grade A and B
areas
• Where installed, design, location, maintenance
• Effective cleanable traps
• Air breaks preventing backflow
• Floor channels open and easily cleanable
9.6.

Sterile Production
Premises
 Changing rooms
– Designed as airlocks
– Effective flushing with filtered air
– Separate rooms for entry and exit desirable
– Hand washing facilities
– Interlocking system for doors
– Visual and/or audible warning system
 Use filtered air supply to maintain pressure cascade
 Pressure differential approximately 10 to 15 Pascales
 Zone of greatest risk – immediate environment

9.7 – 9.9

Sterile Production
Premises
 Pathogenic, highly toxic, radioactive materials
 Pressure cascade may be different
 Decontamination procedures – air, equipment, garments
 Qualification including airflow patterns

– No risk to the product
 Warning system to indicate failure in air supply
 Pressure indicators – results regularly recorded
 Restricted access – e.g. use of barriers 9.9 – 9.12

Sterile Production
Equipment
 Conveyer belts
 Effective sterilization of equipment
 Maintenance and repairs from outside the clean area

– If taken apart, resterilized before use
– Use clean instruments and tools
 Planned maintenance, validation and monitoring

– Equipment, air filtration systems, sterilizers, water
treatment systems 10.1 – 10.5

Sterile Production
Equipment
 Water treatment plants and distribution system

– Design, construction, maintenance
– Operation and design capacity
– Testing programme
 Water for Injection (WFI)

– Produced, stored, distributed – prevention of growth of
microorganisms 10.6
– Constant circulation at temperature above 70, or not more

than 4 degrees Celsius
Sterile Production
Environmental Monitoring - I
Microbiological
 Air samples
 Surface swabs
 Personnel swabs

Sterile Production
Environmental Monitoring - II
Physical
 Particulate matter
 Differential pressures
 Air changes, airflow patterns
 Clean-up time/recovery
 Filter integrity
 Temperature and relative humidity
 Airflow velocity

Sterile Production
Sanitation
 Frequent, thorough cleaning of areas necessary
 Written programme
 Regular monitoring to detect resistant strains of microorganisms
 Chemical disinfection
 Monitoring of disinfectants and detergents
 Dilutions
– Clean containers, stored for defined periods of time 3.1 – 3.2
– Sterilized before use, when used in Grade A or B areas

Sterile Production
Sanitation
 Monitoring of clean areas
 Monitoring of personnel and surfaces after critical operations
 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
 Results considered when batch release is done

3.3

Sterile Production
Sanitation
 Limits of detection established
 Alert and action, and monitoring trends of air quality 3.4
Table 1. Limits for microbial contamination (Information only)
Grade Air sample Settle plates Contact plates Glove print

(CFU/m3) (90mm diameter) (55mm diameter) (5 fingers)
(CFU/4hours) (CFU/plate) (CFU/glove)
A <3 <3 <3 <3
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -

Sterile Production
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
 Special cases
– Supervision in case of outside staff
8.1tissue
– Decontamination procedures (e.g. staff who worked with animal – 8.3
materials)

Sterile Production
Personnel
 High standards of hygiene and cleanliness
 Periodic health checks
 No shedding of particles
 No introduction of microbiological hazards
 No outdoor clothing
 Changing and washing procedure

8.4 – 8.6
 No watches, jewellery and cosmetics

Sterile Production
Personnel
 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
– Grade A and B 8.7
• Headgear, beard and moustache covered, masks, gloves
• No shedding of fibres, and retain particles shed by operators

Sterile Production
Personnel
 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
 Disinfect gloves during operations
 Washing of garments – separate laundry facility
 No damage, and according to validated procedures 8.8 – 8.9

Sterile Production
Group session 1
 You are asked to visit a factory producing the following
product lines:
– Injections in ampoules and vials, including insulin, vaccines and
heat-stable pharmaceuticals
– Sterile eye ointment
 Describe the type of facility you would expect to find
 List the typical rooms, their purpose and air classification

Sterile Production
Possible Issues
 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

Sterile Production
Possible Issues (continued)

 Particulate levels/microorganisms
 Differential pressures
 Air changes
 Temperature/humidity

Sterile Production
Two categories of manufacturing operations:
 Terminally sterilized
– prepared, filled and sterilized
 Aseptic preparation
– some or all stages
1.3

Sterile Production
Manufacture of sterile preparations
 Classification of clean areas
 Manufacturing operation in an appropriate environment cleanliness

level
 Minimize risks – particulate and microbiological contamination –

product and material
 Meet classification "at rest"

– (i.e. "completed installation, equipment installed and operating,
but no operating personnel present").
4.1

Sterile Production
 For sterile pharmaceutical preparations:
 Grade A
– Local zone, high risk operations, e.g. filling, aseptic connections
– Usually UDAF systems used
 Grade B
– Background environment to grade A (in case of aseptic preparation
and filling)
 Grade C and Grade D

4.1
– Clean areas for less critical operations

Sterile Production
Air Classification System
Grade At rest In operation

maximum permitted number of particles/m3
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
3.1

Sterile Production
 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 4.1 – 4.2.

Sterile Production
 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)
4.3 – 4.5

Sterile Production
Airborne particulate classification
WHO GMP US 209E US Customary ISO/TC (209) 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
4.1

Sterile Production
Processing
 Minimise contamination – all stages including before sterilization

and during processing
 No unsuitable materials, e.g. live microbiological organisms
 Minimize activities
– staff movement controlled and methodical
– avoid shedding of particles
 Temperature and humidity comfortable
 Containers and materials in the area

4.15 – 4.16, 4.20 – 4.21

Sterile Production
Processing
 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
– Use appropriate medium/media
– Sufficient number of units - e.g. equal to batch size (small batches)
• acceptable limit
• investigations
– Revalidation: periodic and after change
 New processing procedures validated

– Revalidation after significant changes
– And regular intervals 4.17, 4.18, 4.28

Sterile Production
Processing
 Water sources, water treatment systems and treated water
 Monitored regularly
– Chemicals
– Biological contamination
– Endotoxin
 Water specification
 Records of results and action taken 4.19

Sterile Production
Processing
 Components, bulk product containers and equipment

– fibre generation
– no recontamination after final cleaning
– stage properly identified
– sterilized when used in aseptic areas
 Used in clean areas, passed through double-ended sterilizers or

use triple wrapping 4.22 – 4.23
 Gas used to purge solution or blanket a product – passed

through a sterilizing filter
Sterile Production
Processing
 Bioburden monitored
– Products: Before sterilization
– Working limits established
– Solutions to be filtered before filling (especially LVP)
– Pressure release outlets – hydrophobic microbiological air
filters
 Starting materials – microbiological contamination should be

minimal
4.26, 5.3
 Monitored as per specification
Sterile Production
Processing
 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
4.23 - 4.24

Sterile Production
Group session 2
 Considering the same factory as in the previous group session,

discuss the process of sterilization.
 List all the items that will need to be sterilized (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.

Sterile Production
Possible Issues
 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

Sterile Production
Sterilization
 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
5.1 – 5. 2

Sterile Production
Sterilization
 Validation
– All sterilization processes
– Special attention when non-pharmacopoeia 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 5.4 – 5.5

Sterile Production
Sterilization
 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
5.6 - 5.7

Sterile Production
Sterilization
 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
5.8 - 5.9

Sterile Production
Terminal Sterilization
 Sterilization by heat
 Sterilization by moist heat
 Sterilization by dry heat
 Sterilization by radiation
 Sterilization by gases and fumigants

6

Sterile Production
Sterilization by heat
 Recording of each cycle, e.g. time and temperature chart

– Temperature: validated coolest part
– Check from second independent probe
– Additional chemical or biological indicators
 Heating phase: Sufficient time for the whole load

– Determined for each load
 Cooling phase: After sterilization cycle

– Precautions to prevent contamination
– Sterilized cooling fluid/gas
6.2 – 6.3

Sterile Production
Sterilization by moist heat (heating in an autoclave)
 Water-wetable materials only, and aqueous formulations
 Temperature, time and pressure monitored
 Temperature recorder independent of the controller
 Independent temperature indicator
 Drain – temperature recorded from this position
 Regular leak test when vacuum is part of the cycle
 Material allows for removal of air and penetration of steam
 All parts of the load in contact with steam
 Quality of the steam – no contamination
6.4 – 6.6

Sterile Production
Sterilization by dry heat
 For non-aqueous liquids, dry powders
 Air circulation in the chamber
 Positive pressure in chamber to prevent entry of non-sterile air
 HEPA filtered air supplied
 When removing pyrogens, challenge tests

– validation (using endotoxins)
6.7

Sterile Production
Sterilization by radiation
 Suitable for heat-sensitive materials and products
– confirm suitability of method for material
– ultraviolet irradiation not acceptable
 Contracting service – ensure validation status, responsibilities
 Measurement of dose during procedure
 Dosimeters independent of dose rate
– quantitative measurement
– number, location and calibration time-limit
 Biological indicators only as additional control
 Radiation sensitive colour discs
6.8 – 6.10

Sterile Production
Sterilization by radiation (2)
 Information forms part of the batch record
 Validation to cover effects of variation in density of packages
 Handling procedures to prevent misidentification of irradiated

and non-irradiated materials
 Each package to have a radiation-sensitive indicator
 Total radiation dose administered within a predetermined
period of time
6.10 – 6.13

Sterile Production
Sterilization by gases and fumigants
 Only when no other method is suitable
 e.g. ethylene oxide, hydrogen peroxide vapour
 Validation: Also prove the gas has no damaging effect on product
 Time and conditions for degassing (specified limits) - residue
 Direct contact with microbial cells essential
– Nature and quantity of packaging materials
 Humidity and temperature equilibrium
 Monitoring of each cycle with biological indicators
– time, pressure
– temperature, humidity and gas concentration 6.14 – 6.20

Sterile Production
Sterilization by gases and fumigants (2)
 Post-sterilization storage – controlled manner

– Ventilated conditions
– Defined limit of residual gas
– Validated process
 Safety and toxicity issues
6.21

Sterile Production
Terminally sterilized products

Grade Preparation Remark
D Components and product Ensure low microbial and

particulate count
C Product at unusual risk of e.g. product actively

microbial contamination supports microbial
growth, or
is held for a long period
of time before
sterilization, or
is not prepared mainly in
closed containers
C Filling before sterilization -
4.6 – 4.7

Sterile Production
Terminally sterilized products
A in Filling before sterilization if e.g. slow filling

C background product at unusual risk of operation, or
contamination from Wide neck containers, or
environment Exposure for a few
seconds before sealing
C Preparation and filling Ointments, creams,
suspensions, emulsions
4.8 – 4.9

Sterile Production
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? 7.1 – 7.2

Sterile Production
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

7.3

Sterile Production
Aseptic preparation
D Components after washing

C Preparation of solutions to be
sterile filtered later in the
process
A Preparation and filling of When the product is
(in B sterile ointments, creams, exposed and filtered
background) suspensions, emulsions
4.10, 4.11, 4.14

Sterile Production
Aseptic preparation
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)

4.10 – 4.13
Sterile Production
Sterilization by filtration
 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 before filling - no

fibre shedding or asbestos filters
 Filter integrity testing immediately after use

– also before use if possible
7.4 – 7.7

Sterile Production
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
7.7

Sterile Production
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
7.8 – 7.9

Sterile Production
Quality Control
 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
2.1 -2.2
 Sterility test procedure as per pharmacopoeia, and validated for each product
 Batch processing records, sterility testing records, environmental records
should be reviewed

Sterile Production
Quality Control
 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 2.3

Sterile Production
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 11.1 – 11.3
– results recorded

Sterile Production
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.

Sterile Pharmaceutical Products

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Supplementary Training Modules on

Good Manufacturing Practice

Sterile Pharmaceutical Products

Annex 6. TRS 902, 2002

Sterile | Slide 1 of 62 January 2006

 To review basic GMP requirements in the manufacture of sterile

 To review air classifications for activities related to the manufacture

 To review the different types of sterilization methods

 To review quality assurance aspects in the manufacture and control

 To consider current issues applicable in your country

Sterile | Slide 2 of 62 January 2006

GMP Requirements for Sterile Products

 Specific points relating to minimizing risks of

Sterile | Slide 3 of 62 January 2006

 Production in clean areas

 Appropriate standard of cleanliness

 Filtered air supplied

 Airlocks for entry

 Separate areas for operations

Sterile | Slide 4 of 62 January 2006

 In clean areas, all exposed surfaces:

Sterile | Slide 5 of 62 January 2006

Sterile | Slide 6 of 62 January 2006

 Use filtered air supply to maintain pressure cascade

 Pressure differential approximately 10 to 15 Pascales

 Zone of greatest risk – immediate environment

Sterile | Slide 7 of 62 January 2006

 Pathogenic, highly toxic, radioactive materials

 Pressure cascade may be different

 Decontamination procedures – air, equipment, garments

 Qualification including airflow patterns

 Warning system to indicate failure in air supply

 Pressure indicators – results regularly recorded

 Restricted access – e.g. use of barriers 9.9 – 9.12

Sterile | Slide 8 of 62 January 2006

 Effective sterilization of equipment

 Maintenance and repairs from outside the clean area

 Planned maintenance, validation and monitoring

Sterile | Slide 9 of 62 January 2006

 Water treatment plants and distribution system

 Water for Injection (WFI)

– Constant circulation at temperature above 70, or not more

Sterile | Slide 11 of 62 January 2006

 Air changes, airflow patterns

 Temperature and relative humidity

Sterile | Slide 12 of 62 January 2006

 Frequent, thorough cleaning of areas necessary

 Regular monitoring to detect resistant strains of microorganisms

 Monitoring of disinfectants and detergents

Sterile | Slide 13 of 62 January 2006

 Monitoring of clean areas

 Monitoring of personnel and surfaces after critical operations

 Frequent monitoring in areas where aseptic operations are carried

 Results considered when batch release is done

Sterile | Slide 14 of 62 January 2006

Grade Air sample Settle plates Contact plates Glove print

Sterile | Slide 15 of 62 January 2006

 Minimum number of personnel in clean areas

 Inspections and controls from outside

 Training to all including cleaning and maintenance staff

Sterile | Slide 16 of 62 January 2006

 High standards of hygiene and cleanliness

 Periodic health checks