Sterile Product
• To give an overview of the principles involved in the
manufacture of sterile products
• The overall objective is to produce product that has a
high assurance of sterility (and which meets all other
quality parameters)
• This presentation:
 Summarises the general approach
 Gives a framework for other detailed guides on specific aspects of
sterilisation & sterile manufacturing
 Illustrates the underlying principles
 Provides advice and gives recommendations.
General Principles of Sterile
Moist Heat Sterilization
Dry Heat Sterilization
Aseptic Processing
Environmental Monitoring
Ethylene Oxide Sterilization
Sterile Filtration
Water systems validation
Sterility testing
Radiation Sterilization
Visual Inspection
Sterility is the absence of living organisms
 This is an absolute definition
The probability of achieving sterility depends on the overall process
It is generally accepted that a terminally sterilized product should have a probability
of non-sterility of less than 10-6 (i.e., a lower probability than one in a million of
having a non-sterile unit)
This is often expressed as an SAL Sterility Assurance Level of 106
This is a worst-case figure (with a challenge more resistant than product bioburden).
Real confidence levels are generally very much higher
A figure that has sometimes been quoted for aseptically filled product is probability of
non-sterility of less than 10-3. However, this is harder to analyse as contamination
does not follow a clear statistical distribution. Potential contamination sources are not
randomly distributed.
Why Validate and Control?
• The test for sterility cannot confirm that the whole
batch is sterile
 It is performed on a sample from a batch and has
statistical limitations
 It can miss contamination if only a proportion of units
are non-sterile
• It is thus necessary to recognize and understand
every aspect that could lead to loss of sterility
• Such conditions should be prevented by the
application of carefully designed barriers and/or
control measures.
Development – Validation and
• It is important that the product and process are designed to
maximise sterility assurance
• Wherever possible, the product should be developed to withstand
sterilization in the final container
• Once the product design is defined, a suitable production process
must be developed
• This is installed and validated
• The process must then be tightly controlled to assure reliability and
Product Design Considerations
• For New Products:
Define product and processing requirements
Consider stability of product to the sterilization conditions
Base the process on achieving the required sterility assurance level
Where possible choose terminal sterilization in final container
Define process flow and the important microbiological aspects
Ensure changes are subject to strict change control
• For reviewing existing (marketed) products:
Establish the process description and assess in detail
Preferably, sterilization should be by compendial procedures
Where other procedures are registered, assess SAL
Where necessary (if existing SAL is too low) may need to improve
process and maybe re-register
 Require justification & validation.
Facility Design
• Must be in compliance with company policies and procedures, for
 Must minimise the risk of contamination at all critical stages
 Required Grades of Clean Rooms : need to be appropriate for the
process - e.g. for Terminal Sterilization or Aseptic Fill
Personnel Access and Material Flow
Restricted access, correct gowning
Materials flow, air locks, decontamination, segregation
Segregation/Dedicated HVAC of correct standard
Requires control of Filtration/ΔP/Air Flow/Temp./Pressure/Humidity
Air flow patterns demonstrated
No sinks and drains in Zone A/B areas, air breaks to drains in others
Surfaces and ease of cleaning: smooth unbroken impervious surfaces
Cleaning and disinfection of the
• Cleaning and disinfection is important in environmental control
 Efficacy needs to be validated
 Validated procedures, conducted consistently
• In class A & B areas, the cleaning and disinfectant materials
must be sterilized
 And need to minimise contamination risk in other areas
• Operating procedures must include, at minimum:
 Preparation of cleaning materials (and sterilization if applicable)
 Exact procedure of cleaning & disinfection.
 Responsibility & scheduling.
 Type and concentration of detergents and disinfectants.
 Type of cleaning tools.
• Training is required for cleaning and disinfection of clean rooms
• Routine decontamination using formaldehyde gas should be
• All water systems require good design and validation
• Typically, for pharmacopoeial grades, validation includes
 Two studies over a total of 4 weeks to assess against the acceptance criteria,
 Additional 11 months to verify that the system remains under control
• Must demonstrate consistent production of water of the required
 Physico-chemical,
 Microbiological,
 Biological (endotoxin, where applicable)
• Water systems must be regularly monitored following a defined
written monitoring plan based on results of the validation
Categories of Water
• Water for Injections (WFI)
 For injectables formulation
 Final rinse water for product-contact items (for injectables)
 Freshly prepared or from a validated hot (e.g., >75°C)
storage /distribution system or otherwise protected from
microbial contamination
• Highly Purified Water (HPW)
 To European Pharmacopoeia
• Purified Water (PW)
 For initial washing of product-contact items
 Prepared, suitably stored and distributed to maintain
quality and prevent microbiological proliferation, following
the relevant company procedures.
Gases and Vacuum
• Gases
 Specification equivalent to the room air quality where it is to be used
 In aseptic applications, gases are to be filter sterilized
 Consider sterile filtering non-product contact gases for aseptic
applications. (But, note safety considerations, e.g. avoidance of
 All gas filters to be integrity tested on installation and at defined
• Vacuum Systems
Sometimes used for cleaning and dust control
May be mobile units, fitted with exhaust HEPA filters
Or may have central dust collection
On these, use dedicated vacuum pumps’ protected against back-flow
Design to prevent unprotected route into the aseptic suite.
Equipment (1)
• Equipment Qualification
 To include the critical aspects for sterile product processing
 Qualification of critical aspects of moist heat sterilization, aseptic
processing, dry heat sterilization etc.
• Cleaning and Sanitization of Equipment
 Equipment designed for easy cleaning and sanitization
 For Terminal Sterilization applications, low microbial challenge.
Where possible, critical surfaces should be sterilized
 For aseptic work, the critical (product contact) surfaces must be
sterilized before use. In exceptional cases where this is not
possible (e.g., some stopper bowls), they should be sanitized by
a validated method
 Cleaning validation must show effectiveness and absence of
Equipment (2)
• Equipment Sterilization and handling
 Sterilization must follow a validated procedure
 Aseptic processes designed to minimise aseptic
assembly and intervention
 Unavoidable aseptic assembly needs clear &
precise procedures
 Aseptic assembly must be simulated (worst-case)
in media fill simulation trials
• Sterilization In Place is a good method where
possible – must be validated.
• Training - personnel appropriately trained for sterile processing,
including assessment and documentation:
Basic GMP
Fundamentals of microbiology
Personal hygiene, health and cleanliness
Behaviour and aseptic working techniques
Gowning and entry procedures
Cleaning and disinfection
Sterilization procedures, validation and routine operation
Emergency procedures to protect product quality (e.g. loss of HVAC System, loss of
power, equipment interventions etc.)
• Personnel participating in aseptic processing must have practical
training in aseptic techniques before doing aseptic manipulations
• They must have participated in a successful media fill run.
Gowning and Aseptic Technique
• Gowning
 Personnel must correctly wear appropriate clean room garments
 Detailed, easily understood, gowning procedure (preferably illustrated)
• Aseptic Techniques
 Personnel in the aseptic manufacturing area, must understand the
principles of aseptic procedures
 They must only be considered qualified after appropriate training,
working under supervision and demonstration of competence
 The supervisor should observe technique & correct as necessary
 All personnel directly involved in aseptic processing must participate in a
media fill at least once per year
• Glove disinfection
 Sterile disinfectants must be available (e.g., alcohol based)
 Glove disinfection must be reasonably frequent, defined in SOP.
Environmental Monitoring (1)
• The scope of environmental monitoring
Non-viable particulates,
Viable (microbial) counts
Differential pressures
Air flows
Environmental Monitoring (2)
• Monitoring During Room Qualification
Operational Qualification (OQ) at rest conditions to verify operation
Performance Qualification (PQ) in worst case operational conditions
Action levels should meet USP or Euro GMP as applicable
Alert levels tight enough to detect deterioration, but not so tight that
they become meaningless due to frequent transgression
 PQ must cover a sufficient period to establish consistency
• Routine Monitoring
 Ensures area remains satisfactory. Results should be within alert level
 Results above alert levels need review and perhaps corrective actions
 Above action levels, must trigger appropriate actions (described in
 Results must be assessed for trends so that progressive or sudden
changes in the results may be observed. This should be reviewed
Environmental Monitoring (3)
Deviation Reports and Failure Investigations
The data must be analysed
Where necessary further investigations initiated
Possible contamination sources to be assessed and, eliminated
Outcome and detail must be reported
Physical measurements of the air supply
Physical and microbiological monitoring of the environment
Particles (viable and non-viable) in the air
Micro-organisms settling out of the air
Micro-organisms contaminating surfaces
Presence of micro-organisms on the hands and garments
Recommended Methods for Routine Monitoring
Monitoring Plan
 Defined monitoring plans: tests, locations, alert/action levels & frequencies
 May contain details of water, compressed gas clean steam testing
 A review of environmental data is a requirement for batch release.
Bioburden and Components
• Active Ingredients, Excipients, Additives
 All ingredients should have appropriate biological specifications
 Any limitations to sterilization must be defined
 Description of origin (e.g. virological / prion risk)
• Materials Used in the Process
 Where appropriate, determine bioburden (e.g., ion exchange materials)
• Primary Packaging Components
 Container and the closure and cleaning / sterilization to be clearly
 Steps such as siliconization may need monitoring
 If cleaning/sterilization is by supplier, same exigencies apply
• Container-closure integrity
 The integrity must be validated
 Simulate, where appropriate: stress from processing
 Method appropriate to container/closure system
Weighing, Compounding and
Weighing and compounding must be carried out in suitably classified rooms
Vessels must be cleaned, and sterilized or sanitised as appropriate and
stored dry in a way to prevent microbial contamination
Storage of pre-sterilization intermediates to be controlled & time limited
Following aspects to be considered:
 Pre-filtration bioburden (filter sterilized material)
 Pre-sterilization bioburden
 Appropriate in-process controls
Sterilization of product and product contact materials
 Selection of a suitable sterilization protocol must be based on SAL
 Method must also consider the stability of the product
 Validation always required
 Change control is vital; even apparently minor change must be assessed
Terminal Sterilization
Steam Sterilization
By far the most common method for aqueous-based pharmaceuticals
Preferred cycle is the Pharm Eur reference cycle is 15 minutes at 121°C
The sterilization cycle chosen must be compatible with product stability
Sterilization parameters clearly defined
In conjunction with other controls, the required SAL must be demonstrated
Validation to confirm sterilization conditions consistently throughout the load
Sterilization by Ionizing Radiation
 Common for medical devices, but not for pharmaceuticals.
 Pharm. Eur. reference condition, 25 KiloGray (kGy), has been widely accepted. Other
conditions may be used if validated and accepted by the regulator
 Important to consider susceptibility of the product to radiation damage
Dry Heat Sterilization
 Lower antimicrobial efficacy than moist heat, thus higher temperatures and/or longer
exposures. Pharm Eur reference cycle is 2 hours @ 160°C
 Rarely used for terminal sterilization of pharmaceuticals; in rare cases heat resistant
non-aqueous products may be terminally sterilized.
Sterilization of Items for Aseptic Fill
Steam Sterilization
 Widely used, but careful validation needed – particularly complex items
 Broadly similar to terminal steam sterilization, but two aspects are critical
Quality of saturated steam
Removal of air and subsequent steam penetration
Sterilization by Ionizing Radiation
Dry Heat Sterilization/Depyrogenation
 May be used for temperature sensitive primary packaging or components
 Used for disposables for sterile areas and sterility testing areas
 Validation includes dosimetry, - correct, even, irradiation of the items
 Sterilization/ depyrogenation of heat resistant primary packaging materials
 Pharm Eur notes that temperatures in excess of 220 oC have been frequently used,
the USP suggests 250 ± 15 oC
 Validation must include endotoxin challenge studies
 Dry heat may be used to sterilize non-aqueous preparations (e.g. Ointment bases) at
lower temperature/time relationships, without depyrogenation.
Sterilization of Items for Aseptic Fill
• Ethylene Oxide Sterilization
 Quite widely used to sterilize heat labile components
 European Pharmacopoeia and the European GMP guide indicate
that this method should only be used where there is no suitable
 Hazardous - toxic, potentially carcinogenic, flammable,
potentially explosive
 Generally conducted by specialized contractors
 There are strict regulatory limits on maximum permissible
product residues
 Bulk packs for sterilization must be gas permeable, but sealed
against microbial ingress
 Sterilization must consider packaging, load pattern, gas
penetration (ethylene oxide & water vapour), bulk pack integrity
 Validation and routine monitoring must include Biological
Sterilization by Filtration (Liquids)
 Contaminating organisms are not killed, but are retained on the filters. Any faults in
the filter structure, may compromise this
Validation includes:
 Retention of bacterial challenge: B. diminuta at 107 per cm2
 This is correlated with an integrity test value
Validation should address:
Filter suitability - toxicity, extractables, shedding of particles
Adsorption of product
Compatibility with product solvents
The required filter size and suitability of the filtration equipment
Retention of B.diminuta in the actual product under process conditions
Parameters for the physical integrity test
Routine Filtration
 Conducted in line with the validated parameters
 Check integrity testing, process time, differential pressure, flow rates, sterilization and
reuse of filters.
Performance Qualification of Aseptic
• Based on simulating the risk of contamination in all
aseptic operations
• For a new process, a minimum of three consecutive
satisfactory media filling trials
• For aqueous liquid products, simulation trials use a
liquid microbiological medium
• For solid dosage forms, a powder ‘placebo’ is used,
followed by aseptic reconstitution into a liquid
microbiological medium
• The following slide gives a general overview....
Aseptic Process Simulation
(Media Fill Trial)
• Media Fill Trials (MFTs)
All process stages simulated as closely as possible
Particularly interventions and manual manipulations
Must follow routine procedures and include all interventions
Regular interventions simulated with the same frequency as actual
 In each case, the worst-case eventuality must be covered
 Process must be successfully validated before product filling is
 Revalidation by media fill must be conducted every half year (each line)
• Manufacturing Environment
 Microbiological monitoring must be performed during the trial
• Filling Conditions and Equipment
 All according to routine operating conditions and at normal times of day
 Containers must be passed through all stages.
Thank You
Any Questions

Company name DEPARTMENT Management of GMP