6 February, 2005
VALIDATION OF ASEPTIC PROCESSES
Editor: PIC/S Secretariat
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Table of Contents
1 Introduction. 3
1.1 Purpose........ 3
1.2 Scope........... 3
1.3 General information........ 3
2 Definitions... 4
3 Process Simulation Test Procedures.......... 5
3.1 General Comments........ 5
3.2 Liquid Products.............. 6
3.2.1 Vial Products 6
3.2.2 Sterile Products in Plastic Containers............. 6
3.2.3 Ampoule Products.......... 7
3.3 Injectable Powder Products............. 7
3.4 Suspension Products..... 7
3.5 Freeze Dried (Lyophilized) Products................ 7
3.6 Semi-Solid Products (e.g. sterile ointments).... 7
3.7 Clinical Trials Materials and Small Batch Size Products... 8
3.8 Biological and Biotechnology Products............ 8
3.9 Sterile Bulk Pharmaceuticals.......... 8
4 Process Simulation Test Conditions........... 8
4.1 Test Performance........... 8
4.2 Selection of Growth Medium........... 9
4.3 Incubation Conditions..... 9
4.4 Reading of the Test...... 10
4.5 Test Frequency............ 10
5 Interpretation of Data 10
6 Environmental and Personnel Monitoring. 12
6.1 Air Borne Microbial and Non-Viable Particle Monitoring. 12
6.1.1 Non-viable monitoring.. 12
6.1.2 Microbial Monitoring.... 13
6.2 Intervention Monitoring. 13
7 Staff Training............. 13
8 Important Factors in Validation of Aseptic Manufacturing........ 14
8.1 Container/Closure Integrity Testing................ 14
8.2 Container/Closure Sterilisation..... 14
8.3 Equipment Cleaning and Sterilisation............ 15
8.3.1 Manual cleaning (see PIC/S Document PR 1/99-1, Cleaning validation) and sterilisation. 15
8.3.2 Clean-in-place/sterilise-in-place (CIP/SIP).... 15
8.4 Disinfection. 15
8.5 Filter Validation........... 16
8.6 Vent Filters. 16
8.7 Equipment Maintenance and Testing............. 16
8.8 Blow Fill Seal/Form Fill Seal......... 16
8.9 Sterility Test 17
Authors / working group:
Hansueli Hofstetter, Switzerland
Tobias Gosdschan, Switzerland
Lilian Hamilton, Sweden
Veronika Subai, Hungary
Theo Berg, Netherlands
Paul Hargreaves, United Kingdom
23 January 1997
20 February 1997
17 February 1999
Adoption by the PIC/S Committee
The aim of this document is to provide guidance to the current practice in this field by giving recommendations for the validation of aseptic processes.
This document applies to all manufacturers involved in aseptic processing of finished dosage forms (human and veterinary) as well as manufacturers of sterile labelled
1.3 General information
The basic principles and application of process validation are described in
PIC Document PH 1/96
Validation of aseptic processes relies upon prospective,
Prospective studies would include
the installation ,
Concurrent validation includes a process validation with the same requirements as for prospective studies, but performed during routine production on qualified equipment.
While the r Retrospective validation uses the data of earlier manufactures, but is not a recommended technique for aseptic processes. , process simulation tests include the elements of both prospective and retrospective validation in that results can indicate current and past problems.
Re-validation includes the :
· Regular performance of process simulation studies
· Monitoring of environment, disinfection procedures, equipment cleaning and sterilisation (including containers and closures)
· Routine maintenance and re-qualification of equipment, e.g.
autoclaves, ovens, HVAC (heating, ventilation and air conditioning) systems, water systems, etc.
· Regular integrity testing of
product filters, containers, closures and vent filters
· Re-validation after changes
It is the sum total of all validation data that provides the necessary level of assurance for aseptically produced products.
Validation of aseptic processes includes Process simulation studies (media fills) are simulating simulation of the whole process
in order to evaluate the sterility confidence of the process. Process simulation studies
including include formulation (compounding), filtration and filling with suitable media in order to evaluate the sterility confidence
of the process. Simulations are made to ensure that the regular process for commercial batches repeatedly and reliably produces the finished product of the required quality. However, each process simulation trial is unique and so it is not possible to extrapolate these results directly to actual production contamination rates.
The performance of process simulation tests (media fills) and related test data are only a part of the overall validation strategy for an aseptic process. In addition the following aspects (the most important ones) need to be addressed:
· Container/Closure Integrity Tests
· Container/Closure Sterilisation
· Equipment Cleaning and Sterilisation
· Filter Validation
· Vent Filters
· Equipment Maintenance and Testing
· Blow Fill Seal/Form Fill Seal
· Sterility Test
These subjects are covered under chapter 8: Some other important factors invalidation of aseptic manufacturing.
The methods for simulating an aseptic process vary according to the process used for the various types of products, i.e. liquid, semi-liquid and solid dosage forms.
In thisese Recommendations the term „should“ indicates requirements that are expected to apply unless shown to be inapplicable or replaced by an alternative demonstrated to provide at least an equivalent level of quality assurance.
established microbial or particulate levels giving early warning of potential drift from normal operating conditions which are not necessarily grounds for definitive corrective action but which require follow-up investigation.
operation whereby the product is sterilised separately, then filled and packaged using sterilised containers and closures in critical processing zones.
total number of viable microorganisms on or in pharmaceutical product prior to sterilisation.
a process wherein bulk drug substance is combined with another bulk drug substance and/or one or more excipients to produce a drug product.
defined documented programme which describes the routine particulate and microbiological monitoring of processing and manufacturing areas, and includes a corrective action plan when action levels are exceeded.
test performed to demonstrate that media will support microbial growth.
retentive matrix designed to remove a defined percentage of particulate matter of a defined size.
: Test to determine the functional performance of a filter system.
method of evaluating an aseptic process using a microbial growth medium. (Media fills are understood to be synonymous to simulated product fills, broth trials, broth fills etc.).
scheduled periods of work or production, usually less than 12 hours in length, staffed by alternating groups of workers.
s ampling frequency: e stablished period for collecting samples.
free of any viable organisms. (In practice, no such absolute statement regarding the absence of microorganisms can be proven, see sterilisation.
): Probability that a batch of product is sterile. (SAL is expressed as 10 -n).
s terilisation: v alidated process used to render a product free of viable organisms. Note: In a sterilisation process, the nature of microbiological death of reduction is described by an exponential function.
Therefore, the number of microorganisms which survive a sterilisation process can be expressed in terms of probability. While the probability may be reduced to a very low number, it can never be reduced to zero.
non-shedding porous material capable of removing viable and non-viable particles from gass
3 Process Simulation Test Procedures
3.1 General Comments
Separate dedicated standard equipment should be used exclusively for the preparation of the medium. All aseptic receiving vessels should be covered by a process simulation test on a regular basis unless a validated, pressure hold or vacuum hold test is routinely performed. The media fill should emulate the product fill situation in terms of time taken for filling as well as storage
The following chapter illustrates the test procedures for the various simulation tests for aseptically produced solutions, lyophiles, suspensions, ointments and powders and summarises the considerations to be made.
3.2 Liquid Products
3.2.1 Vial Products
The liquid growth medium for the simulation test is prepared as above and kept in a sterile holding vessel for the maximum permitted holding time before starting the simulation test. If the bulk solution is stored under refrigerated conditions during the holding time then this should also be performed for the medium. Vials and closures should be prepared as in regular production.
3.2.2 Sterile Products in Plastic Containers
Ear and eye drops are typically marketed in plastic containers. Containers, inserts, closures and where applicable overseals are washed and sterilised as in regular production. Instead of sterilisation with heat, irradiation or ethylene oxide are used.
Whilst clear plastic containers are frequently used for process simulation trials, the plastic is usually slightly opaque and thus hinders identification of contaminated units that show only a slight haze. In such case examination under natural or room lighting would not suffice. Where opaque containers are used for process simulation trials the whole contents should be removed for examination.
3.2.3 Ampoule Products
Open or closed ampoule types may be used. They should be sterilised by dry heat and afterwards used in the simulation test as per the regular production run.
Ampoules should be prepared as in regular production.
3.3 Injectable Powder Products
There are two possibilities for simulation of this process. Either by filling a sterilised liquid growth medium into the sterile container or adding a powder (inert or growth medium) before or after a sterile diluent (WFI or growth medium). Inert materials commonly used include: polyethylene glycol 8000 and carboxymethyl cellulose. These materials are usually sterilised by irradiation.
3.4 Suspension Products
This procedure is comparable to the filling of liquid products, except for the process step of maintaining suspension of the ingredients. The stirring or recirculation should be part of the simulation. If aseptic additions are made to the bulk solution these should be simulated by the use of inert sterile liquids/powders.
3.5 Freeze Dried (Lyophilised) Products
The vacuum should be broken with sterile filtered air. Inert gases should not be used as this will prevent the growth of aerobic microorganisms.
Crystallisation of the medium should be prevented because it may reduce the likelihood of recovery of organisms.
3.6 Semi-Solid Products (e.g. sterile ointments)
For this simulation test the liquid growth medium is thickened to the appropriate viscosity, used as in the routine production procedure. Suitable thickening agents are agar and carboxymethyl cellulose. Other agents would need to be validated with regard to lack of their bacteriostatic and fungistatic properties. Metal and plastic ointment tubes prevent the examination of the medium in-situ.
3.7 Clinical Trials Materials and Small Batch Size Products
The size of media fills for small batch size products should at least equal the number of containers filled for the commercial product.
3.8 Biological and Biotechnology Products
The manufacture of these products vary, such that there is not one single process. It may be more practical to validate the various segments of the process individually. The frequency of the revalidation should relate to the one of regular, commercial production.
3.9 Sterile Bulk Pharmaceuticals
Whenever possible a growth medium should be used and the process should be simulated as closely as possible to the normal route of manufacturing the sterile
The aseptic manufacture of a sterile bulk
The validation may include segments, where the use of growth media is not feasible.
4 Process Simulation Test Conditions
4.1 Test Performance
The process simulation test should follow as closely as possible the routine aseptic manufacturing process and include all critical subsequent manufacturing steps. All equipment should remain the same wherever practicable as for the routine process.
Appropriate combinations of container size and opening as well as speed of the processing line should be used (preferably at the extremes).
Worst case conditions are often thought to be the largest container with the widest mouth as it is exposed longer to the environment. However, there are exceptions to this and one of them is small ampoules run at the highest speed as the ampoules may be unstable and cause frequent jams thus necessitating frequent operator intervention.
The process simulation test should represent a „worst case“ situation and include all manipulations and interventions likely to be represented during a shift.
If batches smaller than 3000 units are produced, the minimum number of containers used for the process simulation should be equal to that of the commercial batch size.
Simulation tests should be performed on different days and hours during the week and not only at the beginning of a work day.
Each shift, product type, process - line and container combination should be monitored with process simulation tests at defined intervals (e.g. every 6 months per shift and product line). If the same process is conducted in a separate clean room, this should also be validated.
In order to find the possible source of contamination it may be a good advise to video tape the aseptic fill and also number the individual vials or segregate vials in chronological order during incubation.
4.2 Selection of Growth Medium
The criteria for the selection of growth medium include: low selectivity, clarity, medium concentration and filterability.
Ability to support growth of a wide range of microorganisms: The medium should have a low selectivity i.e. be capable of supporting growth of a wide range of microorganisms such as Bacillus subtilis, Staphylococcus aureus, Candida albicans, Aspergillus niger and Clostridium sporogenes (e.g. Soybean Casein Digest).
The selection of the medium has to be based also on the in house flora (e.g. isolates from monitoring etc.).
In case of nitrogen overlay a medium supporting the growth of anaerobes should be considered.
Growth promotion tests should demonstrate that the medium supports recovery and growth of low numbers of microorganisms, i.e. 10-100 CFU/unit or less.
Growth promotion testing of the media used in simulation studies should be carried out on completion of the incubation period to demonstrate the ability of the media to sustain growth if contamination is present.
Growth should be demonstrated after 5 days at the same incubation temperature as for the simulation test media .
Clarity: The medium should be clear to allow for ease in observing turbidity.
Medium Concentration: Recommendations of the supplier should be followed unless alternative concentrations are validated to deliver equal results.
Filterability: If a filter is used in the aseptic manufacturing process, the medium should be capable of being filtered through the same grade as used in production.
4.3 Incubation Conditions
It is generally accepted to incubate at 20-25°C for a minimum of 14 days without having collected data to support this incubation schedule. It is similarly acceptable for firms who prefer a two temperature incubation schedule to incubate at 20-25°C for a minimum of 7 days followed immediately by incubation at a higher temperature range not to exceed 35°C for a total minimum incubation time of 14 days. Other incubation schedules should be based on supporting data.
Prior to incubation the containers with the microbiological growth medium should be inverted or otherwise manipulated to ensure that all surfaces, included the internal surface of the closure, are thoroughly wetted by the medium. The containers should not be completely filled with medium in order to provide sufficient oxygen for the growth of obligate aerobes. Similarly, containers should not be overlaid with inert gases even though the product may be
The microorganisms present in the containers of the simulation test should be identified to genus but preferably species level to aid determination of the possible sources of the contamination.
4.4 Reading of the Test
When inspecting the containers they should be compared to a known sterile container for comparison as some microbial growth shows up as a faint haze which is difficult to detect unless there is a control container to compare against. Personnel should be trained for this task.
4.5 Test Frequency
A new aseptic processing line or a m ajor and critical change in the aseptic processing line and/or environment should be considered as a „start-up“-validation. Major changes are significant personnel changes (a new shift), modifications in equipment directly in contact with the product, modification in the HVAC system.
For a „start-up“ validation three consecutive satisfactory simulation tests per operator team or shift should be carried out before routine manufacture can start.
The „on-going“ validation should ensure that aseptic conditions are maintained at all times.
If processing lines stand idle for more than six months, this requires a re-validation. Exceeding an action level also demands a re-validation.
5 Interpretation of Data
After the incubation period of the media-filled containers they are visually examined for microbial growth. Contaminated containers should be examined for evidence of container/closure damage which might compromise the integrity of the packaging system. Damaged containers should not be included as failures
Different approaches may be used to determine limits and acceptance criteria.
Ideally the contamination rate should be zero. However currently the accepted contamination rate should be less than 0.1 % with a 95 % confidence
In order to calculate the „worst case“ contamination rate for an observed frequency of failures the following table can be used.