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 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

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 ]dditional rather than replacement

 Specific points relating to minimizing risks of

± microbiological
± particulate matter
± pyrogen

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 Production in clean areas

 ]ppropriate standard of cleanliness

 Filtered air supplied

 ]irlocks for entry

± Personnel and/or equipment
± Materials

 Separate areas for operations

± component preparation (containers and closures)
± product preparation, filling, sterilization, etc. ÷ ÷ ÷

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± ]void 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 M ÷ M 

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 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 ] and B


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 hanging rooms
± uesigned 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 M  M M

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 Pathogenic, highly toxic, radioactive materials

 Pressure cascade may be different

 uecontamination 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 M M M ÷

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 onveyer 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 ÷ ÷ ÷ 

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 Water treatment plants and distribution system

± uesign, construction, maintenance
± Operation and design capacity
± Testing programme

 Water for Injection (WFI)

± Produced, stored, distributed ± prevention of growth of
microorganisms ÷ 

± onstant circulation at temperature above 70, or not more

than 4 degrees elsius
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& #     ' (


 ]ir samples

 Surface swabs

 Personnel swabs

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& #     ' ((

 Particulate matter

 uifferential pressures

 ]ir changes, airflow patterns

 lean-up time/recovery

 Filter integrity

 Temperature and relative humidity

 ]irflow velocity
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 Frequent, thorough cleaning of areas necessary

 Written programme

 Regular monitoring to detect resistant strains of microorganisms

 hemical disinfection

 Monitoring of disinfectants and detergents

± lean containers, stored for defined periods of time ¦ ÷ ¦ 
± Sterilized before use, when used in Grade ] or B areas

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 Monitoring of clean areas

 Monitoring of personnel and surfaces after critical operations

 Frequent monitoring in areas where aseptic operations are carried

± 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


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 Gimits of detection established
 ]lert and action, and monitoring trends of air quality ¦
Table 1. Gimits 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 -
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 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 Î ÷ Î ¦
± uecontamination procedures (e.g. staff who worked with animal
tissue materials)
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 High standards of hygiene and cleanliness

 Periodic health checks

 No shedding of particles

 No introduction of microbiological hazards

 No outdoor clothing

 hanging and washing procedure

Î  Î 
 No watches, jewellery and cosmetics

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 lothing of appropriate quality:

± Grade u
hair, beard, moustache covered
Protective clothing and shoes
± Grade 
Hair, beard, moustache covered
Single or 2-piece suit (covering wrists, high neck), shoes
no fibres to be shed
± Grade ] and B
Headgear, beard and moustache covered, masks, gloves
No shedding of fibres, and retain particles shed by operators
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 Outdoor clothing not in change rooms leading to grade B and 


 hange at every working session, or once a day (if supportive data)

 hange gloves and masks at every working session

 uisinfect gloves during operations

 Washing of garments ± separate laundry facility

 No damage, and according to validated procedures


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 åou 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

 uescribe the type of facility you would expect to find

 Gist the typical rooms, their purpose and air classification

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 Poor design of the building

 Poor design of the systems, e.g. water, HV]

 Flow of personnel

 Flow of material

 No validation or qualification

 Old facilities not complying with current requirements

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!(*  +
 Particulate levels/microorganisms

 uifferential pressures

 ]ir changes


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,      -

 Terminally sterilized
± prepared, filled and sterilized

 ]septic preparation
± some or all stages


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 lassification of clean areas

 Manufacturing operation in an appropriate environment cleanliness


 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").

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 For sterile pharmaceutical preparations:

 Grade ]
± Gocal zone, high risk operations, e.g. filling, aseptic connections
± Usually Uu]F systems used

 Grade B
± Background environment to grade ] (in case of aseptic
preparation and filling)

 Grade  and Grade u ÷

± lean areas for less critical operations

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 . . /  0./  . . /  0./
 1.   1.  
B 1.   1.    
C 1.     1.    
D 1.            


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 To reach Grade B,  and u, the number of air changes should be

appropriate to the size of the area, number of personnel, equipment

 Minimum of 20 air changes per hour

 lean up time about 15-20 minutes

 Good airflow pattern in the area

 HEP]-filtered air

 Suitable methods to determine particulate matter and micro-

± e.g. EU, ISO, Japan, US] ÷ 

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 ontrol particulate during operation

 Monitoring during operation

 ]lert and action limits for particulate and micro

 ]ction taken when exceeded

 ]rea grades should be proven (e.g. validation runs, media fills,

environment, time limits ± based on microbiological
contamination/bioburden found)

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23   4  4      5    
 A 1 .     .  A
 6 1 .     .  6
   . .      7   
 8  .       9   8

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 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

 ontainers and materials in the area

÷ ÷

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 Validation ± should not compromise the processes
 ]septic process validation: Sterile media fill (³broth 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
± Revalidation: periodic and after change

 New processing procedures validated

± Revalidation after significant changes ÷
± ]nd regular intervals
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 Water sources, water treatment systems and treated water

 Monitored regularly
± hemicals
± Biological contamination
± Endotoxin

 Water specification

 Records of results and action taken ÷M

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 omponents, 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  ¦

 Gas used to purge solution or blanket a product ± passed

through a sterilizing filter
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 Bioburden monitored
± Products: Before sterilization
± Working limits established
± Solutions to be filtered before filling (especially GVP)
± Pressure release outlets ± hydrophobic microbiological air

 Starting materials ± microbiological contamination should be


 Monitored as per specification
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  #-%   !:   $

 Washing and drying and sterilization; and sterilization and use

± ]s short as possible
± Time limit validated


 Start of preparation of solution and sterilization (filtration)

± ]s short as possible
± Maximum time set for each product

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 onsidering the same factory as in the previous group session,

discuss the process of sterilization.

 Gist 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

 uiscuss the relevance, need, and the extent of qualification and

validation required.

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 ]utoclave - no pressure gauge

 ]utoclave - no temperature recorder

 ]utoclave - superheated steam

 lean room - pressure differentials

 Exposure for settle plates

 Interlocks turned off

 Rusty Gaminar airflow cabinets

 HEP] filters not checked regularly

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 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

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± ]ll 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:
± ]ll parts of the load
± Each type of load
± Physical measurements and biological indicators (where
± Verified at least annually and after change
± Records maintained    

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 For effective sterilization:

 Whole of the material subjected to the treatment

 Biological indicators:

 ]dditional method of monitoring

 Storage and use, quality checked through positive control

 Risk of contamination
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 uifferentiation between sterilized and not-yet-sterilized

 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  Î  M

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 Sterilization by heat

 Sterilization by moist heat

 Sterilization by dry heat

 Sterilization by radiation

 Sterilization by gases and fumigants

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Sterilization by heat

 Recording of each cycle, e.g. time and temperature chart

± Temperature: validated coolest part
± heck from second independent probe
± ]dditional chemical or biological indicators

 Heating phase: Sufficient time for the whole load

± uetermined for each load

 ooling phase: ]fter sterilization cycle

± Precautions to prevent contamination
± Sterilized cooling fluid/gas
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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

 urain ± 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

 ]ll parts of the load in contact with steam

 Quality of the steam ± no contamination    

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Sterilization by dry heat

 For non-aqueous liquids, dry powders

 ]ir circulation in the chamber

 Positive pressure in chamber to prevent entry of non-sterile air

 HEP] filtered air supplied

 When removing pyrogens, challenge tests

± validation (using endotoxins)

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Sterilization by radiation
 Suitable for heat-sensitive materials and products
± confirm suitability of method for material
± ultraviolet irradiation not acceptable
 ontracting service ± ensure validation status, responsibilities
 Measurement of dose during procedure
 uosimeters independent of dose rate
± quantitative measurement
± number, location and calibration time-limit
 Biological indicators only as additional control
 Radiation sensitive colour discs  Î  ÷

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Sterilization by radiation (2)
 Information forms part of the batch record

 Validation to cover effects of variation in density of

 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  ÷  ÷¦

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Sterilization by gases and fumigants
 Only when no other method is suitable
 e.g. ethylene oxide, hydrogen peroxide vapour
 Validation: ]lso prove the gas has no damaging effect on product
 Time and conditions for degassing (specified limits) - residue
 uirect 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
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Sterilization by gases and fumigants (2)

 Post-sterilization storage ± controlled manner

± Ventilated conditions
± uefined limit of residual gas
± Validated process

 Safety and toxicity issues


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]    ;  !  

]septic processing

 Objective is to maintain the sterility of a product,

assembled from sterile components

 Operating conditions so as to prevent microbial


 What do you think are the aspects that require careful

attention?  ÷  

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]    ;  !  

]septic processing (2)

 areful attention to:



 ritical surfaces

 ontainer/closure sterilization

 Transfer procedures

 Maximum holding period before filling ¦

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÷ ÷¦

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 Through a sterile filter of 0,22 µm or less, into previously sterilized

± remove bacteria and moulds
± not all viruses or mycoplasmas

 onsider complementing with some degree of heat treatment

 uouble 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

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 ;  !<   * +
 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

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 ;  !<   * +
 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

 Î  M

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= % 
 Samples for sterility testing should be representative
 From parts of the batch, most at risk
± ]septic filling ± at beginning and end of batch filling, and after
± 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 eachproduct

 Batch processing records, sterility testing records, environmental records

should be reviewed

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= % 

 Endotoxin testing for injectable products
± Water for injection, intermediate and finished product

 ]lways for large volume infusion solutions

 Pharmacopoeia method, validated for each product

 Failure of the test ± investigation

 orrective action ¦

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 ontainers 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
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 onsidering the same factory as in the previous group
sessions, devise a plan for monitoring of the facility.

 Gist the parameters to be tested, tests to be used,

acceptance criteria and frequency of testing.

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