• 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:
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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.

• 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 10 6
• 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.

• The test for sterility cannot confirm that the whole batch is sterile
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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 assurance
• Such conditions should be prevented by the application of carefully designed barriers and/or control measures.

• 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 consistency.

• For New Products:
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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:
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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.

• Must be in compliance with company policies and procedures, for example:
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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
HVAC-System
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 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:
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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.
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• Training is required for cleaning and disinfection of clean rooms
• Routine decontamination using formaldehyde gas should be avoided.

• All water systems require good design and validation
• Typically, for pharmacopoeial grades, validation includes
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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 quality
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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 studies.

• Water for Injections (WFI)
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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)
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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
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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 leakage)
All gas filters to be integrity tested on installation and at defined intervals
• Vacuum Systems
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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.

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To include the critical aspects for sterile product processing
Qualification of critical aspects of moist heat sterilization, aseptic processing, dry heat sterilization etc.
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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 residues.

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• Training - personnel appropriately trained for sterile processing, including assessment and documentation:
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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
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Personnel must correctly wear appropriate clean room garments
Detailed, easily understood, gowning procedure (preferably illustrated)
• Aseptic Techniques
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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
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Sterile disinfectants must be available (e.g., alcohol based)
Glove disinfection must be reasonably frequent, defined in SOP.

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• Monitoring During Room Qualification
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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
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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 guide),
Results must be assessed for trends so that progressive or sudden changes in the results may be observed. This should be reviewed regularly.

• Deviation Reports and Failure Investigations
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The data must be analysed
Where necessary further investigations initiated
Possible contamination sources to be assessed and, eliminated
Outcome and detail must be reported 
• Recommended Methods for Routine Monitoring
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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 
• Monitoring Plan
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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.

• Active Ingredients, Excipients, Additives
 All ingredients should have appropriate biological specifications
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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 specified
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Steps such as siliconization may need monitoring
If cleaning/sterilization is by supplier, same exigencies apply
• Container-closure integrity
 The integrity must be validated
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Simulate, where appropriate: stress from processing
Method appropriate to container/closure system

• 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:
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Pre-filtration bioburden (filter sterilized material)
Pre-sterilization bioburden
 Appropriate in-process controls
• Sterilization of product and product contact materials
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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

• Steam Sterilization
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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
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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
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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.

• Steam Sterilization
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Widely used, but careful validation needed – particularly complex items
Broadly similar to terminal steam sterilization, but two aspects are critical
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Quality of saturated steam
Removal of air and subsequent steam penetration
• Sterilization by Ionizing Radiation
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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 
• Dry Heat Sterilization/Depyrogenation
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Sterilization/ depyrogenation of heat resistant primary packaging materials
Pharm Eur notes that temperatures in excess of 220 o C have been frequently used, the USP suggests 250 ± 15 o C
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.

• Ethylene Oxide Sterilization
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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 alternative
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 indicators.

• Principle:
 Contaminating organisms are not killed, but are retained on the filters. Any faults in the filter structure, may compromise this
• Validation includes:
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Retention of bacterial challenge: B. diminuta at 107 per cm 2
This is correlated with an integrity test value
• Validation should address:
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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.

• 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....

• Media Fill Trials (MFTs)
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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 process
In each case, the worst-case eventuality must be covered 
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Process must be successfully validated before product filling is permitted
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
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All according to routine operating conditions and at normal times of day
Containers must be passed through all stages.