EUROPEAN COMMISSION
ENTERPRISE AND INDUSTRY DIRECTORATE-GENERAL
Consumer goods
Pharmaceuticals
Brussels, 25 November 2008 (rev.)
EudraLex
The Rules Governing Medicinal Products in the European Union
Volume 4
EU Guidelines to
Good Manufacturing Practice
Medicinal Products for Human and Veterinary Use
Annex 1
Manufacture of Sterile Medicinal Products
(corrected version)
Document History
Previous version dated 30 May 2003, in operation since September 2003
Revision to align classification table of clean rooms, to include
guidance on media simultations, bioburden monitoring and capping
of vials
November 2005 to
December 2007
Date for coming into operation and superseding 01 March 20091
Please note correction on the implementation of provisions for capping of vials!
1 Note: Provisions on capping of vials should be implemented by 01 March 2010.
Commission Européenne, B-1049 Bruxelles / Europese Commissie, B-1049 Brussel – Belgium. Telephone: (32-2) 299 11 11
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ANNEX 1
MANUFACTURE OF STERILE MEDICINAL PRODUCTS
Principle
The manufacture of sterile products is subject to special requirements in order to minimize
risks of microbiological contamination, and of particulate and pyrogen contamination. Much
depends on the skill, training and attitudes of the personnel involved. Quality Assurance is
particularly important, and this type of manufacture must strictly follow carefully established
and validated methods of preparation and procedure. Sole reliance for sterility or other quality
aspects must not be placed on any terminal process or finished product test.
Note:
This guidance does not lay down detailed methods for determining the microbiological and
particulate cleanliness of air, surfaces etc. Reference should be made to other documents such
as the EN/ISO Standards.
General
1. The manufacture of sterile products should be carried out in clean areas entry to which
should be through airlocks for personnel and/or for equipment and materials. Clean areas
should be maintained to an appropriate cleanliness standard and supplied with air which has
passed through filters of an appropriate efficiency.
2. The various operations of component preparation, product preparation and filling should be
carried out in separate areas within the clean area. Manufacturing operations are divided into
two categories; firstly those where the product is terminally sterilised, and secondly those
which are conducted aseptically at some or all stages.
3. Clean areas for the manufacture of sterile products are classified according to the required
characteristics of the environment. Each manufacturing operation requires an appropriate
environmental cleanliness level in the operational state in order to minimise the risks of
particulate or microbial contamination of the product or materials being handled.
In order to meet “in operation” conditions these areas should be designed to reach certain
specified air-cleanliness levels in the “at rest” occupancy state. The “at-rest” state is the
condition where the installation is installed and operating, complete with production
equipment but with no operating personnel present. The “in operation” state is the condition
where the installation is functioning in the defined operating mode with the specified number
of personnel working.
The “in operation” and “at rest” states should be defined for each clean room or suite of clean
rooms.
For the manufacture of sterile medicinal products 4 grades can be distinguished.
Grade A: The local zone for high risk operations, e.g. filling zone, stopper bowls, open
ampoules and vials, making aseptic connections. Normally such conditions are provided by a
laminar air flow work station. Laminar air flow systems should provide a homogeneous air
speed in a range of 0.36 – 0.54 m/s (guidance value) at the working position in open clean
room applications. The maintenance of laminarity should be demonstrated and validated.
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A uni-directional air flow and lower velocities may be used in closed isolators and glove
boxes.
Grade B: For aseptic preparation and filling, this is the background environment for the grade
A zone.
Grade C and D: Clean areas for carrying out less critical stages in the manufacture of sterile
products.
Clean room and clean air device classification
4. Clean rooms and clean air devices should be classified in accordance with EN ISO 14644-
1. Classification should be clearly differentiated from operational process environmental
monitoring. The maximum permitted airborne particle concentration for each grade is given
in the following table.
Maximum permitted number of particles per m3 equal to or greater
than the tabulated size
At rest In operation
Grade 0.5 µm 5.0µm 0.5 µm 5.0µm
A 3 520 20 3 520 20
B 3 520 29 352 000 2 900
C 352 000 2 900 3 520 000 29 000
D 3 520 000 29 000 Not defined Not defined
5. For classification purposes in Grade A zones, a minimum sample volume of 1m
3
should be
taken per sample location. For Grade A the airborne particle classification is ISO 4.8 dictated
by the limit for particles ≥5.0 µm. For Grade B (at rest) the airborne particle classification is
ISO 5 for both considered particle sizes. . For Grade C (at rest & in operation) the airborne
particle classification is ISO 7 and ISO 8 respectively. For Grade D (at rest) the airborne
particle classification is ISO 8. For classification purposes EN/ISO 14644-1 methodology
defines both the minimum number of sample locations and the sample size based on the class
limit of the largest considered particle size and the method of evaluation of the data
collected.
6. Portable particle counters with a short length of sample tubing should be used for
classification purposes because of the relatively higher rate of precipitation of particles
≥5.0µm in remote sampling systems with long lengths of tubing. Isokinetic sample heads
shall be used in unidirectional airflow systems.
7. “In operation” classification may be demonstrated during normal operations, simulated
operations or during media fills as worst-case simulation is required for this. EN ISO 14644-2
provides information on testing to demonstrate continued compliance with the assigned
cleanliness classifications.
Clean room and clean air device monitoring
8. Clean rooms and clean air devices should be routinely monitored in operation and the
monitoring locations based on a formal risk analysis study and the results obtained during the
classification of rooms and/or clean air devices.
9. For Grade A zones, particle monitoring should be undertaken for the full duration of critical
processing, including equipment assembly, except where justified by contaminants in the
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process that would damage the particle counter or present a hazard, e.g. live organisms and
radiological hazards. In such cases monitoring during routine equipment set up operations
should be undertaken prior to exposure to the risk. Monitoring during simulated operations
should also be performed. The Grade A zone should be monitored at such a frequency and
with suitable sample size that all interventions, transient events and any system deterioration
would be captured and alarms triggered if alert limits are exceeded. It is accepted that it may
not always be possible to demonstrate low levels of ≥5.0 µm particles at the point of fill when
filling is in progress, due to the generation of particles or droplets from the product itself.
10. It is recommended that a similar system be used for Grade B zones although the sample
frequency may be decreased. The importance of the particle monitoring system should be
determined by the effectiveness of the segregation between the adjacent Grade A and B zones.
The Grade B zone should be monitored at such a frequency and with suitable sample size that
changes in levels of contamination and any system deterioration would be captured and
alarms triggered if alert limits are exceeded.
11. Airborne particle monitoring systems may consist of independent particle counters; a
network of sequentially accessed sampling points connected by manifold to a single particle
counter; or a combination of the two. The system selected must be appropriate for the particle
size considered. Where remote sampling systems are used, the length of tubing and the radii
of any bends in the tubing must be considered in the context of particle losses in the tubing.
The selection of the monitoring system should take account of any risk presented by the
materials used in the manufacturing operation, for example those involving live organisms or
radiopharmaceuticals.
12. The sample sizes taken for monitoring purposes using automated systems will usually be a
function of the sampling rate of the system used. It is not necessary for the sample volume to
be the same as that used for formal classification of clean rooms and clean air devices.
13. In Grade A and B zones, the monitoring of the ≥5.0 µm particle concentration count
takes on a particular significance as it is an important diagnostic tool for early detection of
failure. The occasional indication of ≥5.0 µm particle counts may be false counts due to
electronic noise, stray light, coincidence, etc. However consecutive or regular counting of
low levels is an indicator of a possible contamination event and should be investigated. Such
events may indicate early failure of the HVAC system, filling equipment failure or may also
be diagnostic of poor practices during machine set-up and routine operation.
14. The particle limits given in the table for the “at rest” state should be achieved after a
short “clean up” period of 15-20 minutes (guidance value) in an unmanned state after
completion of operations.
15. The monitoring of Grade C and D areas in operation should be performed in accordance
with the principles of quality risk management. The requirements and alert/action limits will
depend on the nature of the operations carried out, but the recommended “clean up period”
should be attained.
16. Other characteristics such as temperature and relative humidity depend on the product and
nature of the operations carried out. These parameters should not interfere with the defined
cleanliness standard.
17. Examples of operations to be carried out in the various grades are given in the table below
(see also paragraphs 28 to 35):
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Grade Examples of operations for terminally sterilised products. (see paragraphs 28-
30)
A Filling of products, when unusually at risk
C Preparation of solutions, when unusually at risk. Filling of products
D Preparation of solutions and components for subsequent filling
Grade Examples of operations for aseptic preparations. (see paragraphs. 31-35)
A Aseptic preparation and filling.
C Preparation of solutions to be filtered.
D Handling of components after washing.
18. Where aseptic operations are performed monitoring should be frequent using methods
such as settle plates, volumetric air and surface sampling (e.g. swabs and contact plates).
Sampling methods used in operation should not interfere with zone protection. Results from
monitoring should be considered when reviewing batch documentation for finished product
release. Surfaces and personnel should be monitored after critical operations. Additional
microbiological monitoring is also required outside production operations, e.g. after
validation of systems, cleaning and sanitisation.
19. Recommended limits for microbiological monitoring of clean areas during operation:
Recommended limits for microbial contamination (a)
Grade air sample
cfu/m3
settle plates
(diameter 90 mm)
cfu/4 hours (b)
contact plates
(diameter 55 mm)
cfu/plate
glove print
5 fingers
cfu/glove
A < 1 < 1 < 1 < 1
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -
Notes
(a) These are average values.
(b) Individual settle plates may be exposed for less than 4 hours.
20. Appropriate alert and action limits should be set for the results of particulate and
microbiological monitoring. If these limits are exceeded operating procedures should
prescribe corrective action.
Isolator technology
21. The utilisation of isolator technology to minimize human interventions in processing areas
may result in a significant decrease in the risk of microbiological contamination of aseptically
manufactured products from the environment. There are many possible designs of isolators
and transfer devices. The isolator and the background environment should be designed so that
the required air quality for the respective zones can be realised. Isolators are constructed of
various materials more or less prone to puncture and leakage. Transfer devices may vary from
a single door to double door designs to fully sealed systems incorporating sterilisation
mechanisms.
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22. The transfer of materials into and out of the unit is one of the greatest potential sources of
contamination. In general the area inside the isolator is the local zone for high risk
manipulations, although it is recognised that laminar air flow may not exist in the working
zone of all such devices.
23. The air classification required for the background environment depends on the design of
the isolator and its application. It should be controlled and for aseptic processing it should be
at least grade D.
24. Isolators should be introduced only after appropriate validation. Validation should take
into account all critical factors of isolator technology, for example the quality of the air inside
and outside (background) the isolator, sanitisation of the isolator, the transfer process and
isolator integrity.
25. Monitoring should be carried out routinely and should include frequent leak testing of the
isolator and glove/sleeve system.
Blow/fill/seal technology
26. Blow/fill/seal units are purpose built machines in which, in one continuous operation,
containers are formed from a thermoplastic granulate, filled and then sealed, all by the one
automatic machine. Blow/fill/seal equipment used for aseptic production which is fitted with
an effective grade A air shower may be installed in at least a grade C environment, provided
that grade A/B clothing is used. The environment should comply with the viable and non
viable limits at rest and the viable limit only when in operation. Blow/fill/seal equipment used
for the production of products which are terminally sterilised should be installed in at least a
grade D environment.
27. Because of this special technology particular attention should be paid to, at least the
following:
• equipment design and qualification
• validation and reproducibility of cleaning-in-place and sterilisation-in-place
• background clean room environment in which the equipment is located
• operator training and clothing
• interventions in the critical zone of the equipment including any aseptic assembly
prior to the commencement of filling.
Terminally sterilised products
28. Preparation of components and most products should be done in at least a grade D
environment in order to give low risk of microbial and particulate contamination, suitable for
filtration and sterilisation. Where the product is at a high or unusual risk of microbial
contamination, (for example, because the product actively supports microbial growth or must
be held for a long period before sterilisation or is necessarily processed not mainly in closed
vessels), then preparation should be carried out in a grade C environment.
29. Filling of products for terminal sterilisation should be carried out in at least a grade C
environment.
30. Where the product is at unusual risk of contamination from the environment, for example
because the filling operation is slow or the containers are wide-necked or are necessarily
exposed for more than a few seconds before sealing, the filling should be done in a grade A
zone with at least a grade C background. Preparation and filling of ointments, creams,
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suspensions and emulsions should generally be carried out in a grade C environment before
terminal sterilisation.
Aseptic preparation
31. Components after washing should be handled in at least a grade D environment. Handling
of sterile starting materials and components, unless subjected to sterilisation or filtration
through a micro-organism-retaining filter later in the process, should be done in a grade A
environment with grade B background.
32. Preparation of solutions which are to be sterile filtered during the process should be done
in a grade C environment; if not filtered, the preparation of materials and products should be
done in a grade A environment with a grade B background.
33. Handling and filling of aseptically prepared products should be done in a grade A
environment with a grade B background.
34. Prior to the completion of stoppering, transfer of partially closed containers, as used in
freeze drying should be done either in a grade A environment with grade B background or in
sealed transfer trays in a grade B environment.
35. Preparation and filling of sterile ointments, creams, suspensions and emulsions should be
done in a grade A environment, with a grade B background, when the product is exposed and
is not subsequently filtered.
Personnel
36. Only the minimum number of personnel required should be present in clean areas; this is
particularly important during aseptic processing. Inspections and controls should be
conducted outside the clean areas as far as possible.
37. All personnel (including those concerned with cleaning and maintenance) employed in
such areas should receive regular training in disciplines relevant to the correct manufacture of
sterile products. This training should include reference to hygiene and to the basic elements of
microbiology. When outside staff who have not received such training (e.g. building or
maintenance contractors) need to be brought in, particular care should be taken over their
instruction and supervision.
38. Staff who have been engaged in the processing of animal tissue materials or of cultures of
micro-organisms other than those used in the current manufacturing process should not enter
sterile-product areas unless rigorous and clearly defined entry procedures have been followed.
39. High standards of personal hygiene and cleanliness are essential. Personnel involved in
the manufacture of sterile preparations should be instructed to report any condition which
may cause the shedding of abnormal numbers or types of contaminants; periodic health
checks for such conditions are desirable. Actions to be taken about personnel who could be
introducing undue microbiological hazard should be decided by a designated competent
person.
40. Wristwatches, make-up and jewellery should not be worn in clean areas.
41. Changing and washing should follow a written procedure designed to minimize
contamination of clean area clothing or carry-through of contaminants to the clean areas.
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42. The clothing and its quality should be appropriate for the process and the grade of the
working area. It should be worn in such a way as to protect the product from contamination.
43. The description of clothing required for each grade is given below:
• Grade D: Hair and, where relevant, beard should be covered. A general protective suit
and appropriate shoes or overshoes should be worn. Appropriate measures should be
taken to avoid any contamination coming from outside the clean area.
• Grade C: