products

1. STOP
Annex 6. TRS 902, 2002
Basic Principles of GMP
Sterile Pharmaceutical Products

2. STOP
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

3. STOP
Sterile Production
GMP Requirements for Sterile Products
 Additional rather than replacement
 Specific points relating to minimizing risks of contamination
ä microbiological
ä particulate matter
ä pyrogen

4. STOP
Sterile Production
General Considerations
 Production in clean areas
 Appropriate standard of cleanliness
 Filtered air supplied
 Airlocks for entry
ä personnel and/or equipment
ä materials
 Separate areas for operations
ä component preparation (containers and closures)
ä product preparation
ä filling, sterilization, etc. 1.1 – 1-2

5. STOP
9.1 – 9.6
Sterile Production
Premises
 Design
ä avoid unnecessary entry of supervisors and control personnel
ä operations observed from outside
 In clean areas, all exposed surfaces
ä smooth, impervious, unbroken
ä minimize shedding and accumulation of particles,
microorganisms
ä permit cleaning and disinfection
ä no uncleanable recesses, ledges, shelves, cupboards,
equipment
ä sliding doors undesirable
ä false ceilings sealed

6. STOP
9.6.
Sterile Production
Premises (continued)
 In clean areas, all exposed surfaces (2)
ä proper installation of pipes and ducts, no recesses,
no unsealed openings
ä 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

7. STOP
9.7 – 9.9
Sterile Production
Premises (continued)
 Changing rooms
ä designed as airlocks
ä effective flushing with filtered air
ä separate rooms for entry and exit desirable
ä hand washing facilities
ä interlocking system for doors
ä visual and/or audible warning system
 Use filtered air supply to maintain pressure cascade
 Pressure differential approximately 10 to 15 Pascals
 Zone of greatest risk – immediate environment

8. STOP
9.9 – 9.12
Sterile Production
Premises (continued)
 Pathogenic, highly toxic, radioactive materials
 Pressure cascade may be different
 Decontamination procedures – air, equipment, garments
 Qualification including airflow patterns
ä 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. STOP
10.1 – 10.5
Sterile Production
Equipment
 Conveyer belts
 Effective sterilization of equipment
 Maintenance and repairs from outside the clean area
ä if taken apart, resterilized before use
ä use clean instruments and tools
 Planned maintenance, validation and monitoring
ä equipment, air filtration systems, sterilizers, water
treatment systems

10. STOP
10.6
Sterile Production
Equipment (continued)
 Water treatment plants and distribution system
ä design, construction, maintenance
ä operation and design capacity
ä testing programme
 Water for Injection (WFI)
ä produced, stored, distributed – prevention of growth
of microorganisms
ä constant circulation at temperature above 70, or not
more than 4 degrees Celsius

11. STOP
Sterile Production
Environmental Monitoring - I
Microbiological
 Air samples
 Surface swabs
 Personnel swabs

12. STOP
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

13. STOP
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
ä clean containers, stored for defined periods of time
ä Sterilized before use, when used in Grade A or B
areas
3.1 – 3.2

14. STOP
Sterile Production
Sanitation (continued)
 Monitoring of clean areas
 Monitoring of personnel and surfaces after critical operations
 Frequent monitoring in areas where aseptic operations are
carried out
ä settle plates, volumetric air samples, surface
sampling (swabs and contact plates)
ä sampling methods should not contaminate the area
 Results considered when batch release is done
3.3

15. STOP
Sterile Production
Sanitation (continued)
 Limits of detection established
 Alert and action, and monitoring trends of air quality
Table 1. Limits for microbial contamination (information only)
Grade Air sample
(CFU/m3)
Settle plates
(90mm diameter)
(CFU/4hours)
Contact plates
(55mm diameter)
(CFU/plate)
Glove print
(5 fingers)
(CFU/glove)
A < 3 < 3 < 3 < 3
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -
3.4

16. STOP
8.1 – 8.3
Sterile Production
Personnel
 Minimum number of personnel in clean areas
ä especially during aseptic processing
 Inspections and controls from outside
 Training to all including cleaning and maintenance staff
ä initial and regular
ä manufacturing, hygiene, microbiology
 Special cases
ä supervision in case of outside staff
ä decontamination procedures (e.g. staff who worked
with animal tissue materials)

17. STOP
8.4 – 8.6
Sterile Production
Personnel (continued)
 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
 No watches, jewellery and cosmetics

18. STOP
8.7
Sterile Production
Personnel (continued)
 Clothing of appropriate quality:
ä Grade D
– hair, beard, moustache covered
– protective clothing and shoes
ä Grade C
– hair, beard, moustache covered
– single or 2-piece suit (covering wrists, high neck), shoes
– no fibres to be shed
ä Grade A and B
ä headgear, beard and moustache covered, masks, gloves
ä not shedding fibres, and retain particles shed by operators

19. STOP
8.8 – 8.9
Sterile Production
Personnel (continued)
 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

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

21. STOP
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

22. STOP
Sterile Production
Possible Issues (continued)
 Particulate levels/microorganisms
 Differential pressures
 Air changes
 Temperature/humidity

23. STOP
Sterile Production
Two categories of manufacturing operations
 Terminally sterilized
ä prepared, filled and sterilized
 Aseptic preparation
ä some or all stages
1.3

24. STOP
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"
ä (That is "completed installation, equipment
installed and operating, but no operating
personnel present") 4.1

25. STOP
Sterile Production
Manufacture of sterile preparations
 For sterile pharmaceutical preparations:
 Grade A
ä local zone, high risk operations, e.g. filling, aseptic
connections
ä usually UDAF systems used
 Grade B
ä background environment to Grade A (in case of
aseptic preparation and filling)
 Grade C and Grade D
ä Clean areas for less critical operations
4.1

26. STOP
Air Classification System
Sterile Production
Grade At rest In operation
maximum permitted number of particles/m3
0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm > 5 µ
A 3 500 0 3 500 0
B 3 500 0 350 000 2 000
C 350 000 2 000 3 500 000 20 000
D 3 500 000 20 000 not defined not defined
3.1

27. STOP
Sterile Production
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
ä e.g. EU, ISO, Japan, USA 4.1 – 4.2.

28. STOP
Sterile Production
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)
4.3 – 4.5

29. STOP
Airborne particulate classification
Sterile Production
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

30. STOP
4.15 – 4.16, 4.20 – 4.21
Sterile Production
Processing
 Minimize contamination - all stages including before
sterilization and during processing
 No unsuitable materials, e.g. live microbiological organisms
 Minimize activities
ä staff movement controlled and methodical
ä avoid shedding of particles
 Temperature and humidity comfortable
 Containers and materials in the area

31. STOP
4.17, 4.18, 4.28
Sterile Production
Processing
 Validation – should not compromise the processes
 Aseptic process validation: sterile media fill (“broth fills”)
ä simulate actual operation – intimate as closely as
possible
ä simulate worst expected condition
ä use appropriate medium/media
ä sufficient number of units, e.g. equal to batch size
(small batches)
– acceptable limit
– investigations
ä revalidation: periodic and after change
 New processing procedures validated
ä revalidation after significant changes
ä and regular intervals

32. STOP
4.19
Sterile Production
Processing
 Water sources, water treatment systems and treated water
 Monitored regularly
ä chemicals
ä biological contamination
ä endotoxin
 Water specification
 Records of results and action taken

33. STOP
4.22 – 4.23
Sterile Production
Processing
 Components, bulk product containers and equipment
ä fibre generation
ä no recontamination after final cleaning
ä stage properly identified
ä sterilized when used in aseptic areas
 Used in clean areas, passed through double-ended sterilizers
or use triple wrapping
 Gas used to purge solution or blanket a product – passed
through a sterilizing filter

34. STOP
4.26, 5.3
Sterile Production
Processing
 Bioburden monitored
ä products: before sterilization
ä working limits established
ä solutions to be filtered before filling (especially
LVP)
ä pressure release outlets – hydrophobic
microbiological air filters
 Starting materials – microbiological contamination should be
minimal
 Monitored as per specification

35. STOP
4.23 - 4.24
Sterile Production
Processing
 Time intervals: components, bulk containers,
equipment
 Washing and drying and sterilization; and sterilization and
use
ä as short as possible
ä time limit validated
 Time intervals: product
 Start of preparation of solution and sterilization (filtration)
ä as short as possible
ä maximum time set for each product

36. STOP
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

37. STOP
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

38. STOP
5.1 – 5. 2
Sterile Production
Sterilization
 Methods of sterilization
ä moist or dry heat
ä irradiation (ionizing radiation)
ä sterilizing gaseous agents (e.g. ethylene oxide)
ä filtration with subsequent aseptic filling
 Whenever possible: terminal sterilization by heat in their
final container - method of choice

39. STOP
5.4 – 5.5
Sterile Production
Sterilization
 Validation
ä all sterilization processes
ä special attention when non-pharmacopoeial methods
are used
ä non-aqueous or oily solutions
 Before the method is adopted – its suitability and efficacy
demonstrated with desired conditions
ä all parts of the load
ä each type of load
ä physical measurements and biological indicators
(where appropriate)
ä verified at least annually and after change
ä records maintained

40. STOP
5.6 - 5.7
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

41. STOP
5.8 - 5.9
Sterile Production
Sterilization
 Differentiation between sterilized and not-yet-sterilized
products
 Each basket/tray or other carrier, properly labelled
ä name of material
ä batch number
ä sterilization status
 Use of autoclave tape
 Sterilization records for each run – approved as part of the
batch release procedure

42. STOP
6
Sterile Production
Terminal Sterilization
 Sterilization by heat
 Sterilization by moist heat
 Sterilization by dry heat
 Sterilization by radiation
 Sterilization by gases and fumigants

43. STOP
6.2 – 6.3
Sterile Production
Terminal Sterilization
Sterilization by heat
 Recording of each cycle, e.g. time and temperature chart
ä temperature: validated coolest part
ä check from second independent probe
ä additional chemical or biological indicators
 Heating phase: sufficient time for the whole load
ä determined for each load
 Cooling phase: after sterilization cycle
ä precautions to prevent contamination
ä sterilized cooling fluid/gas

44. STOP
6.4 – 6.6
Sterile Production
Terminal Sterilization
Sterilization by moist heat (heating in an autoclave)
 Water-wettable 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

45. STOP
6.7
Sterile Production
Terminal Sterilization
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
ä validation (using endotoxins)

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

47. STOP
6.10 – 6.13
Sterile Production
Terminal Sterilization
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

48. STOP
6.14 – 6.20
Sterile Production
Terminal Sterilization
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
ä nature and quantity of packaging materials
 Humidity and temperature equilibrium

49. STOP
6.21
Sterile Production
Terminal Sterilization
 Monitoring of each cycle with biological indicators
ä time, pressure
ä temperature, humidity
ä gas concentration
Sterilization by gases and fumigants (2)
 Post-sterilization storage – controlled manner
ä ventilated conditions
ä defined limit of residual gas
ä validated process
 Safety and toxicity issues

50. STOP
Sterile Production
Terminally sterilized products
Grade Preparation Remark
D Components and product Ensure low microbial and
particulate count
C Product at unusual risk of
microbial contamination
E.g. product actively
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

51. STOP
Sterile Production
Terminally sterilized products
Grade Preparation Remark
A in
C background
Filling before sterilization if
product at unusual risk of
contamination from
environment
E.g. slow filling
operation, or
Wide neck containers, or
Exposure for a few
seconds before sealing
C Preparation and filling Ointments, creams,
suspensions, emulsions
4.8 – 4.9

52. STOP
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

53. STOP
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

54. STOP
Sterile Production
Aseptic preparation
Grade Preparation Remark
D Components after washing
C Preparation of solutions to be
sterile filtered later in the
process
A
(in B
background)
Preparation and filling of
sterile ointments, creams,
suspensions, emulsions
When the product is
exposed and filtered
4.10, 4.11, 4.14

55. STOP
Sterile Production
Aseptic preparation
Grade Preparation Remark
A
(in B
background)
Sterile starting materials and
components
(Unless subjected to
sterilization or filtration
through a microorganism
retaining filter later in the
process)
A
(in B
background)
Preparation of solutions (if
not to be sterile filtered later)
A
(in B
background)
Handling and filling of
aseptically prepared
products,
A
(in B
background)
Handling of exposed sterile
equipment
A
(in B
background)
Transfer of partially closed
containers, before complete
stoppering
E.g. in freeze drying
(Grade B environment if
in sealed transfer trays)
4.10 – 4.13

56. STOP
Sterile Production
Sterilization by Filtration
 Through a sterile filter of 0,22 µm or less, into previously
sterilized containers
ä remove bacteria and moulds
ä 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
ä also before use if possible 7.4 – 7.7

57. STOP
Sterile Production
Sterilization by Filtration (2)
 Validation to include
ä time taken to filter a known volume
ä 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

58. STOP
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.
ä removal of ingredients
ä releasing substances into product
7.8 – 7.9

59. STOP
Sterile Production
Quality Control
 Samples for sterility testing should be representative
 From parts of the batch, most at risk
ä aseptic filling - at beginning and end of batch
filling, and after interruptions
ä heat sterilized – coolest part of the load
 Sterility of the batch ensured through validation
ä validated sterilization cycle
ä media fill
 Sterility test procedure as per pharmacopoeia, and validated
for each product
 Batch processing records, sterility testing records,
environmental records should be reviewed 2.1 -2.2

60. STOP
Sterile Production
Quality Control
 Endotoxin testing for injectable products
ä 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

61. STOP
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
ä illumination and background
ä eyesight checks of operators
ä allowed frequent breaks
 Other methods
ä validated, and equipment performance checked at
intervals
ä results recorded
11.1 – 11.3

62. STOP
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