A critical look at Annex 1:

Manufacture of Sterile
Medicinal Products
Good Medical Practices
June 2018

1
EMA published the draft for the new Annex 1
“Manufacture of Sterile Medicinal Products” at The new document will have 11 sections:
December 20, 2017.
1. Scope
The guideline has significantly grown, from 16 to at least 50 pages 2. Principle
now in draft.
3. Pharmaceutical Quality System (PQS)
The document has now much more in common with the 4. Personnel
WHO Annex 6 (WHO good manufacturing practices for sterile 5. Premises
pharmaceutical products) and the PIC/S Annex 1 (Manufacture of 6. Equipment
sterile medicinal products) but comprises much more details (WHO 7. Utilities
and PIC/S guidelines have respectively 24 and 17 pages). 8. Production and specific technologies
Techniques like Blow Fill Seal and others are discussed in the
9. Viable and non-viable environmental and process
comprehensive chapter 8 “production and specific Technologies”.
monitoring
Chapters about Premises and Equipment are more detailed, while the 10. Quality control (QC)
chapter about Utilities is completely new. 11. Glossary

2
 1. Scope (new)

The scope makes clear that the manufacture of sterile medicinal products covers a wide
range of types, batch sizes, processes, primary packaging materials and technologies.
The guidance should be used for all sterile medicinal products and sterile active
substances, to ensure that microbial, particulate and pyrogen contamination associated
with microbes is prevented in the final product.

Important is the second paragraph of the scope: the intent of

the Annex is to provide guidance for sterile medicinal products.
However, some of the principles and guidance may be used to
support the manufacture of other products that are not intended
to be sterile (such as certain liquids, creams, ointments and low
bioburden biological intermediates) but where the control of
microbial, particulate and pyrogen contamination, to reduce it as far
as possible, is considered important.

3
2. Principle
Principle in the current Annex 1 is short: requirements are needed in
order to minimize risks of microbiological, particulate and pyrogen
contamination. Training and attitudes of the personnel are needed,
QA is particularly important: 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.
The draft Annex 1 explains much more.
In order to minimize risks of microbiological, particulate and
pyrogen contamination, following key areas should be considered:
a) Optimization, qualification and validation according to annex11
and annex 15 is needed for facility, equipment and process
design. The use of appropriate current technologies should be
implemented to ensure protection and control of the product
b) Personnel must have appropriate skills, training and attitudes
(also stipulated in the current Annex 1)
c) Very similar to the two points above: design, commissioning,
qualification and monitoring of the process and monitoring
systems must be done by personnel with appropriate
knowledge
Processes, equipment, facilities and manufacturing activities should
be managed in accordance with QRM principles.
Strange is the next sentence: Risk assessments should be used to
justify alternative approaches to those specified in this Annex only
if these alternative approaches meet or surpass the intent of this
Annex.
This means you need risk assessments, even if you follow, or even do
more than what is written in the Annex? Shortly said: you simply need
risk assessment for everything you do, even if you surpass the annex.
Also the new Annex stresses out the importance of Quality
Assurance. New, but again, very logical:
A contamination control strategy should be implemented across
the facility in order to assess the effectiveness of all the control and
monitoring measures employed. This assessment should lead to
corrective and preventative actions being taken as necessary.
The next sentence should be evident in every modern QRM: The
strategy should consider all aspects of contamination control and
its life cycle with ongoing and periodic review and update of the
strategy as appropriate.
Personal opinion: The principle in the new Annex has grown
significantly, with a lot more of explanations, but is this all necessary?
For example, in the current annex, you only find: The manufacture
of sterile products is subject to special requirements in order to
minimize risks of microbiological contamination, and of particulate
and pyrogen contamination.
If you want to minimize the risk of microbiological contamination,
it is logical you have a contamination control strategy, no? And
everything else that is being told, is common sense in every modern
quality system:
• effectiveness of this control should be assessed
• the assessment should lead to corrective and preventive actions
• all aspects of contamination control and its life cycle should be
considered
• periodic review and update is needed.
After the explanation of the QRM parts of the contamination control
strategy, the new annex again tells us that thorough technical and
process knowledge is necessary, and learns us that microbiological
and cellular debris (pyrogens and endotoxins) as well as particulate
matter are potential sources of contamination.
4

What follows is an enumeration of elements to be
considered within a documented contamination control
strategy:
a) Design of both the plant and process.
b) Equipment and facilities.
c) Personnel.
d) Utilities.
e) Raw Materials Control – including in-process
controls.
f) Product containers and closures.
g) Vendor approval – such as key component suppliers,
sterilization of components and single use systems,
and services.
h) For outsourced services, such as sterilization,
sufficient evidence should be provided to the
contract giver to ensure the process is operating
correctly.
i) Process risk assessment.
j) Process validation.
k) Preventative maintenance – maintaining equipment
and premises (planned and unplanned maintenance)
to a standard that will not add significant risk of
contamination.
l) Cleaning and disinfection.
m) Monitoring systems - including an assessment of the
feasibility of the introduction of scientifically sound,
modern methods that optimize the detection of
environmental contamination.
n) Prevention – Trending, investigations, corrective and
preventive actions (CAPA), root cause determination
and the need for more robust investigational tools.
o) Continuous improvement based on information from
the above systems.
Again, nothing new, all of this is discussed in other parts of the
Eudralex volume 4.
Another way of looking at this way of working in the new
Annex: Annex 1 can be seen as a standalone document with
all necessary information about the manufacture of sterile
medicinal products. A thorough knowledge of all other
Eudralex documents is not necessary, and as a plus, by
repeating everything again here, you have no excuse at all of
not knowing, and not complying!
The principle part ends with two notes:
Note 1 is the same as in the current guideline: guidance regarding
detailed methods for determining microbiological and particulate
cleanliness should be taken from the ISO standards and
pharmacopoeial monographs.
Note 2 says that additional guidance regarding the preparation
of unlicensed sterile medicinal products normally performed by
healthcare establishments for direct supply to patients can be found
in Annex 1: “Guidelines on the standards required for the sterile
preparation of medicinal products” of PIC/S.
“The principle in the new Annex has grown
significantly, with a lot more of explanations,
but is this all necessary?
For example, in the current annex, you only find:
The manufacture of sterile products is subject to
special requirements in order to minimize risks of
microbiological contamination, and of particulate
and pyrogen contamination.”
If you want to minimize the risk of microbiological
contamination, it is logical you have a
contamination control strategy, no? And everything
else that is being told, is common sense in every
modern quality system:
• effectiveness of this control should be assessed
• the assessment should lead to corrective and
preventive actions
• all aspects of contamination control and its
life cycle should be considered
• periodic review and update is needed.
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3. Pharmaceutical Quality System (PQS) (new)
In addition to the PQS requirements detailed in chapter 1 of the EU GMPs, some extra
guidance is given, because of the complexity of the manufacture of sterile medicinal
products.

Next to chapter 1, the PQS for sterile product manufacturers should ensure that:
a) A risk management system is integrated into the product life cycle to Minimise microbiological
contamination. Safety, quality and efficacy of the product and assurance of sterility must be
assured.
b) Knowledge and expertise is needed
c) The importance of root cause analysis of procedures, process or equipment failure is repeated
d) Also, the importance of a documented and regularly reviewed risk assessment in relation to
contamination prevention and the sterile process is stressed out again.
e) Storage conditions should be compliant with registration, and pose no risk to contamination or
container integrity.
f) Persons responsible for the quality release must have appropriate knowledge and access to all
relevant information

Investigations should be performed for non-conformities such as sterility test failures or environmental
monitoring excursions, with a focus on potential impact to sterility. A thorough impact assessment is
needed, with reasoning why specific batches are included or excluded from the investigation.

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4. Personnel

The beginning of chapter 2 – Personnel of the Eudralex is repeated: there should be

sufficient, qualified and experienced personnel for the manufacture and testing of
sterile medicines.

Just as in the current guideline, there is told that a minimum of personnel should be present in
cleanrooms, and inspections and controls should be done outside as much as possible.
New is that the maximum of operators in critical areas should be determined based on QRM,
documented and validated.

Training needs are more excessive: training should include reference to:
• hygiene A lot of extra (and new) is written for personnel
• cleanroom practices working in a grade A/B cleanroom:
• contamination control
• aseptic techniques The personnel working in a grade A/B cleanroom should
• potential safety implications to the patient of a loss of product be trained for aseptic gowning and aseptic practices.
sterility Compliance with aseptic gowning procedures should be
• basic elements of microbiology assessed and confirmed and this should be periodically
Next to trained, operators should be qualified and assessed. Based on reassessed at least annually and should involve both visual
this assessment and trending, a system for disqualification of personnel and microbiological assessment (using additional locations
must be in place, after witch a full retraining and requalification is such as arms and chest). Only trained personnel who have
needed. passed the gowning assessment and have participated in
a successful aseptic process simulation (APS) test, during
which they performed their normal duties, should be
authorized to enter any grade A/B area, in which aseptic
operations will be conducted, or are being conducted,
whilst unsupervised. The microbial monitoring of personnel
in the grade A/B area should be performed to assess their
aseptic behaviour. This monitoring should take place
immediately after completion of a critical intervention and
upon each exit from the cleanroom. It should be noted that
there should also be an ongoing continuous monitoring
program for personnel including some consideration of
periodic monitoring under the supervision of the quality
unit.
Written procedures are needed which describe the access
of outside staff in A/B rooms who did not receive above
training. This access should only be given in exceptional
circumstances.

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Mostly unchanged: the high standards of personal hygiene and
cleanliness of personnel that are essential. Jewellery and make-up is
still not allowed in clean areas, mobile phones are added to the list.
Staff who have been engaged in the processing of other tissue
materials or of cultures of micro-organisms, need rigorous, clearly
defined and effective entry procedures before entering sterile
product areas.
Changing and hand washing is, of course, kept. Already done in
practice but know clearly described in the general changing part:
Garments should be visually checked for cleanliness and integrity
prior to entry to the clean room. For sterilized garments, particular
attention should be taken to ensure that garments and eye
coverings have been sterilized and that their packaging is integral
before use. Reusable garments should be replaced based at a set
frequency determined by qualification or if damage is identified.
This means mirrors should be available in changing areas!
Not much changed to the clothing requirements in
each grade. Most striking: the moustache should also be
covered in grade D, before, this was only described from
grade C, while beard had to be covered as from grade D.
Shoes should be disinfected, in all grades.
New requirement in grade A/B: Garments should be packed
and folded in such a way as to allow operators to change into
the garments with contact to the outer surfaces of the garment
reduced to a minimum.
Two good practices that a lot of companies already used, did find its
way in the guideline:
• It is recommended that facility suits, including dedicated socks
be worn before entry to change rooms for grade C and B.
Where clothing is reused this should be considered as part of
the qualification.
• Activities in clean areas, especially when aseptic operations
are in progress, should be kept to a minimum and movement
of personnel should be controlled and methodical to avoid
excessive shedding of particles and organisms due to over-
vigorous activity. Operators performing aseptic operations
should adhere to strict aseptic technique at all times. To
prevent changes in air currents that introduce lower quality air,
movement adjacent to the critical area should be restricted and
the obstruction of the path of the unidirectional airflow must
be avoided. The ambient temperature and humidity should be
set to prevent shedding due to operators becoming too cold
(leading to excessive movement) or too hot.
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5. Premises

A short general overview states that the manufacture of sterile products should be
carried out in clean areas, with airlocks for personnel entry and/or for equipment and
materials. Air supply must pass through filters of appropriate efficiency.

Grade A: Extra in the grade A description is air measurement: the
point at which the air speed is measured should be clearly justified
in the protocol. As a plus, air visualization studies are necessary to
demonstrate air movement that supports protection of the product
and open components with unidirectional air at the working height.
Entry into the grade A area by operators should be minimized by
facility, process and procedural design.
The extra comment in the former Annex 1 version “A uni-directional
air flow and lower velocities may be used in closed isolators and
glove boxes.”, is left out in the new draft.
Grade B: extra: In general, only grade C cleanrooms should interface
with the grade B aseptic processing area. Lower grades can be
considered where isolator technology is used (refer to clause 338
5.19-5.20).

Guidelines on clean areas are given, such as:

• Materials liable to generate fibers should not be permitted in clean areas
• False ceilings should be designed and sealed to prevent contamination from the space above them
• Sinks and drains should be prohibited in grade A/B areas. In other areas air breaks should be fitted between the machine or sink
and the drains. Floor drains in lower grade rooms should be fitted with traps or water seals to prevent back flow and should be
regularly cleaned and disinfected.
• Airlocks should be designed and used to provide physical separation and to minimize microbial and particulate contamination
of the different areas, and should be present for material and personnel moving from different grades, typically airlocks used for
personnel movement are separate to those used for material movement. They should be flushed effectively with filtered air. The
final stage of the airlock should, in the at-rest state, be the same grade as the area into which it leads. The use of separate changing
rooms for entering and leaving clean areas is generally desirable.
Both airlock doors should not be opened simultaneously. For airlocks leading to grade A and B, an interlocking system should be
used.
o Personnel airlocks. A cascade concept should be followed for personnel (e.g. from grade D to grade C to grade B). In general
hand washing facilities should be provided only in the first stage of the changing rooms.
o Material airlocks: material should pass through hatches with active filtered air, any risk of contamination by incoming material
or by entering air must be avoided.
All materials and equipment that enters A and B areas, must be part of the qualification list.
• A HEPA or ULPA filtered air supply should maintain a positive pressure and an air flow relative to surrounding areas of a lower grade
under all operational conditions and should flush the area effectively. Adjacent rooms of different grades should have a pressure
differential of 10 - 15 Pascals (guidance values).
• Air flow patterns should be visualised in grade A/B areas to evaluate if airflow is unidirectional. Where unidirectional air flow is not
demonstrated, corrective actions, such as design improvements, should be implemented.
• A warning system should be provided to indicate failure in the air supply and reduction of pressure differentials below set limits.
Indicators of pressure differences should be fitted between areas, based on QRM principles. These pressure differences should be
recorded regularly or otherwise documented.
• Consideration should be given to designing facilities that permit observation of activities from outside the clean areas, e.g. through
the provision of windows or remote camera access with a complete view of the area and processes to allow observation and
supervision without entry.

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Barrier technologies
Isolator (current annex) is called RABS (restricted access barrier
system) in the new annex 1.
Most guidance regarding RABS is the same as the isolator guidance
before, and rather general:
• RABS is used to minimize human interventions
• After sterilization, transfer of material into the RABS must be
avoided
• Appropriate validation, both of the RABS and its sterilization
process, is needed
• Regular integrity and leak tests must be performed (new: using
visual, mechanical and physical methods)
Furthermore, everything depends on the specific RABS, so
everything must be reasoned with risk assessments:
• Background B is needed, but a minimum of background D can
be sufficient for open, positive pressure isolators or closed
isolators with decontamination by a sporicidal agent.
• All activities, materials, transfer, decontamination, disinfection
and sterilization, air quality, … shall be considered for the design
of the RABS.
• The critical zone should meet grade A with unidirectional air
flow, but turbulent air flow can be justified.
A positive air flow must be ensured, but a negative can be
justified is containment of product is considered essential
Clean room and clean air device qualification
Qualification should be done according to Annex 15 of EU GMP. ISO
14644 series are given as reference for classification.
For classification, the airborne particles equal to or greater than
0.5 µm should be measured. In the table for maximum permitted
airborne particle concentration during classification, only the
maximum of particles equal to or greater than 0.5 µm are given.
Non-viable monitoring
During qualification Only particles equal to or greater than 0.5 µm
are considered
During environmental monitoring Same limits as during qualification for particles
equal to or greater than 0.5 µm, also particles
equal to or greater than 5 µm must be
monitored
This is contrast with the current guideline, which gives also a
maximum for particles equal to or greater than 5 µm per m³.
A clear explanation is given for the “in operation” and “at rest” states.
For sampling locations, reference is made to the ISO, however, a
higher number of samples and sample volume is typically required
for the aseptic processing room and the immediately adjacent
environment (grade A/B).
For later stages of qualification and classification, such as
performance qualification, locations should be based on a
documented risk assessment and knowledge of the process and
operations to be performed in the area.
The limits for microbial contamination in operation are kept
identical. Individual settle plates may be exposed for less than 4
hours, but (New): if they are exposed for less than 4 hours the
limits in the table should still be used, no recalculation is necessary.
(Remark): Shouldn’t the less than 4 hours be more specified to less
than, but close to 4 hours?
During critical operations, settle plates should be exposed for the
duration of critical operations, and changed after 4 hours.
(New): It is emphasized that the results for grade A should be 0 cfu
recovered, any recovery should result in an investigation.
Qualification and classification of clean rooms should be clearly
differentiated from operational process environmental monitoring.
Characteristics such as temperature and relative humidity depend
on the product and operations, but should not interfere with the
defined cleanliness.
(NEW): Requalification for grade A en B zones is necessary every
6 months, for C and D every 12 months. Requalification is also
necessary after changes to equipment, facility or processes, based
on QRM principles.
Tables are given in this section (qualification of cleanrooms), similar
tables are repeated in the Environmental monitoring section.
The difference between these tables:
Viable monitoring
Air samples, settle plates and contact plates
must be used
The same, but also glove prints are monitored
(this means that glove print is not used during
qualification of A and B environment, which is
rather logical, since no manufacturing takes
place at that time).
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Disinfection
Finally, the premises part is concluded with some guidances about the disinfection of clean areas. In
comparison with the old part of sanitation, extra recommendations are given, most of which is well
known to industry.:
• A written disinfection programme is needed, which effectiveness should be assessed, including
the detection of resistant or spore forming strains
• Surface contamination must be performed first for disinfection to be effective
• More than one type of disinfecting agent should be employed, including a sporicidal agent
• Effectiveness of the disinfectants during their shelf life must be shown, specific to the facilities,
equipment and processes they are used in
Microbial contamination of disinfectants and detergents should be monitored, when used in
grade A and B, they should be sterile prior to use.
• Cleaning programs should be effective in the removal of disinfectant residues
• Fumigation or vapour disinfection of clean areas such as Vapour Hydrogen Peroxide (VHP) may
be useful for inaccessible places

11
6. Equipment

The old equipment part dealt both with equipment and water systems.
In the new version, the equipment part has grown, and the more extensive
recommendations for water systems are moved to the new Utilities part.

New:
• Written, detailed description of the equipment design should be produced (including diagrams
as appropriate) and kept up to date. It should describe the product and other critical gas and fluid
pathways and controls in place.
• Equipment monitoring requirements should be determined during qualification. Process alarm events
should be reviewed, approved and evaluated for trends.
• The cleaning process should be validated so that it can be demonstrated that it:
o Can remove any residues that would otherwise create a barrier between the sterilizing agent and
the equipment surfaces.
o Prevents chemical and particulate contamination of the product during the process and prior to
disinfection.
• All critical surfaces that come into direct contact with sterile materials should be sterile.
• Already in the current guide, and kept almost identical in the new:
• Conveyor belt should not pass through a partition between A or B area, unless it’s continually sterilized

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7. Utilities (new)
The current Annex speaks about water treatment plants (constructed to have water of an appropriate
quality) and Water for injections (produced, stored and distributed to prevent microbial growth) in one
paragraph.
The new Annex has more than two pages, covering utilities general, and speaks more specific about:
• Water systems
• Steam used for sterilization
• Compressed gases and vacuum systems
• Cooling systems
General
All already well known to industry:
• High risk utilities
o should be more thorough controlled than others, with
high risk utilities being those that:
––Directly contact the product
––Contact materials that will become part of the product
––Control contamination of surfaces that contact the
product
––Otherwise directly contact the product
o Should be regularly trended for the critical parameters
• Utility functions should be as expected
• Installation of pipes and ducts should not create recesses,
unsealed openings and surfaces which are difficult to clean
• Drawings should be available, showing pipeline flow, slopes
diameter and length; tanks; valves; filters; drains and sampling
points
The last point is important. Most companies for sure have drawings,
but maybe not with all details described here.
Water systems
Again, quite some well-known guidances:
• Water treatment should minimize the risk of microbial
contamination and proliferation, water produced should comply
with the current monograph of the relevant pharmacopeia
• Flow should remain turbulent through the pipes to prevent
microbial adhesion
• Sloping of piping to provide complete drainage and avoiding of
dead legs monitoring and maintenance of filters in the system
• A predetermined schedule of sterilization or disinfection or
regeneration to prevent the formation of biofilms
when disinfecting with chemicals, a validated rinsing procedure
should follow
• Sampling schedule should ensure representative water samples
analysis on a regular basis
However, with for some companies probably less known, and (very)
strict extra’s:
• Validation of the water system must take seasonal variation into
account
• Sterilization or disinfection or regeneration is needed after
microbial count exceed action and alert limits.
After disinfection, water should be analyzed and results should
be approved before use (does this mean a stop in production
during/after every disinfection cycle of the loop, as long as
approval is not given?)
• Sampling, monitoring and trending should be performed at a
specified interval including all outlets and user points (already
done in industry), but:
A sample from the worst case sample point, e.g. the end of the
distribution loop return, should be included each time the water
is used for manufacturing and manufacturing processes.
Please update your procedures, to be sure they include: A
breach of an alert limit should trigger review and follow-up,
which might include investigation and corrective action.
Any breach of an action limit should lead to a root cause
investigation and risk assessment.
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WFI (water for injections) should be produced from purified water,
stored and distributed so microbial growth is prevented. How WFI
should be produced is not completely clear, the next sentence
in the draft should be rewritten: Where the WFI is produced
by methods other than distillation further techniques post
Reverse osmosis (RO) membrane should be considered such as
nanofiltration, and ultra-filtration.
Hydrophobic bacteria retentive vent filters at WFI storage tanks
should be sterilized and integrity tested, before and after use.
WFI sytems should be monitored continuous (for TOC and
conductivity).
Steam used for sterilization
The water quality for the pure steam generator must be minimum
purified water.
Steam should be of suitable quality, without additives which could
cause contamination of product or equipment.
Quality of the steam must be assessed against parameters as:
• Non-condensable gases
• Dryness value (dryness fraction)
• Superheat
• Steam condensate quality
Compressed gases and vacuum systems
Direct contact compressed gases should be of appropriate quality,
and comply with the pharmacopoeial monographs, if applicable.
At point of use, the gases must be filter sterilized (with pore size max
0.22 µm).
Important: The confirmation of the filter integrity should be part of
the batch release process.
Backflow should be prevented when vacuum or pressure systems
are shut off.
Cooling systems
Rules about the cooling system address possible contamination:
• If much as possible hydraulic and cooling systems should be
kept out of the filling room
if this is not possible, controls to are necessary
• Leaks from the cooling system must be detectable (indication
system needed)
• Leak testing should be done periodically and after maintenance
• Periodic cleaning and disinfection of vacuum system and
cooling systems are necessary
Note the difference (new) for qualification and
monitoring: for qualification, only particles ≥ 0.5 µm
are in scope, for monitoring, also particles ≥ 5 µm
must me measured.
14
8. Production and Specific Technologies
Most of the subparts in this chapter were already discussed in the current Annex, but
everything is discussed in more detail. This chapter takes 17 pages, which is more than
the current Annex 1, which has only 15 pages in total.

Remarkable: in the current Annex, British spelling is used for Examples of operations and which grades they should be performed in:
sterilisation, while in the new Annex, the European Union chooses to
follow the American spelling, and writes sterilization. A ––
Critical processing zone.
––
Aseptic assembly of filling equipment.
––
Aseptic connections (should be sterilized by steam-in-
Terminally sterilized products
place whenever feasible).
Nothing new here, the only extra guidance in the new guideline says ––
Aseptic compounding and mixing.
that processing of bulk solution should include a filtration step to ––
Replenishment of sterile product, containers and
reduce bioburden prior to filling into the final product containers. closures.
––
Removal and cooling of items from heat sterilizers.
A table is added, which summarizes the text, and gives examples
––
Staging and conveying of sterile primary packaging
of which operations should be performed (for terminally sterilized
components.
products) in which grade:
––
Aseptic filling, sealing, transfer of open or partially
stoppered vials, including interventions.
A ––
Loading and unloading of a lyophilizer
Filling of products, when unusually at risk.
C B
Preparation of solutions, when unusually at risk. Filling of ––
Direct support zone for the critical processing (grade
products. A) zone.
––
Transport and preparation of packaged equipment,
D Preparation of solutions and components for subsequent components and ancillary items for introduction into
filling. the grade A zone.
––
Removal of sealed product from the grade A zone.
C ––
Preparation of solutions to be filtered.
Aseptic preparation
Completely different compared with the current chapter, which D ––
Cleaning of equipment.
only tells us in which grades which processes should be done. This ––
Handling of components, equipment and accessories
is also present in the new one, but more comprehensive, and again, after washing.
starting with a table that gives a handy overview. ––
Assembly of cleaned equipment to be sterilized.
The new annex starts with explaining what aseptic preparation is,
Extra comments and clarifications on the table are given.
and explains the necessity of defining the aseptic process and the
risks and control strategy associated with it. Again, it is stipulated
that microbiological, pyrogen and particulate contamination must
be minimised (Here they use British spelling again) during the
complete production cycle.
Where possible, RABS, isolators or closed systems should be
considered.

15
The chapter ends with a logical (aseptic manipulations
should be minimized, and I’ll stop commenting on the
spelling) and a new and important part:
The duration of each aspect of the aseptic manufacturing process
should be limited to a defined and validated maximum, including:
a) Time between equipment, component, and container cleaning,
drying and sterilization.
b) Holding time for sterilized equipment, components, and
containers prior to and during filling/assembly.
c) The time between the start of the preparation of a solution and
its sterilization or filtration through a micro-organism-retaining
filter. There should be a set maximum permissible time for
each product that takes into account its composition and the
prescribed method of storage.
d) Aseptic assembly.
e) Holding sterile product prior to filling.
f) Filling.
g) Maximum exposure time of sterilized containers and closures in
the critical processing zone (including filling) prior to closure.
Finishing of sterile products
Everything of the current guideline is kept, with some extra
guidances in the new annex:
• Visual inspection alone is not considered as an acceptable
integrity test method
• Transportation/shipping requirements should be taken into
consideration for container closure integrity validation
• Manual capping must be performed in grade A conditons with B
background (not really new, is already explained above)
• In the case where capping is conducted as a clean process with
grade A air supply protection, vials with missing or displaced
stoppers should be rejected prior to capping. Appropriately
validated, automated methods for stopper height detection
should be in place. Microbial ingress studies (or alternative
methods) should be utilized to determine the acceptable
stopper height displacement.
• Where human intervention is required at the capping station,
appropriate technology should be used to prevent direct
contact with the vials
• Again, the use of RABS and isolators is promoted
• Batches with unusual levels of defects, when compared
to routine defect levels for the process, should lead to
investigation and consideration of partial or the whole rejection
of the batch concerned.
A defect library should be generated and maintained which
captures all known defects.
There is a clear difference in the operator qualification. The current
annex says: “Operators doing the inspection should pass regular
eye-sight checks, with spectacles if worn, and be allowed
frequent breaks from inspection.”
In the new Annex, this is: “ Operators performing the inspection
should undergo robust visual inspection qualification (whilst wearing
corrective lenses, if these are normally worn) at least annually.”
But it does not stop here:
“The qualification should be undertaken using appropriate sample
sets and taking into consideration worst case scenarios (e.g.
inspection time, line speed (where the product is transferred to the
operator by a conveyor system), component size or fatigue at the
end of shift) and should include consideration of eyesight checks.
Operator distractions should be removed and frequent breaks of
appropriate duration from inspection should be taken.”
Already done in industry, and now also part of the new annex:
• Automated methods of inspection should be validated
• Results of the inspection should be recorded and defect types
and levels trended.
Actually very understandable, but probably new (and strict) for some
companies:
Reject rates for the various defect types should also be trended.
Investigations should be performed as appropriate to address
adverse trends or discovery of new defect types. Impact to product
on the market should be assessed as part of this investigation.
Sterilization
This part starts (new) with telling that finished product should be
terminally sterilized, as this provides a greater assurance of sterility
than a sterilizing filtration process or aseptic processing.
Where this is not possible, consideration should be given to using
terminal bioburden reduction steps, such as heat treatments,
combined with aseptic processing.
QRM is necessary also for selection, design and location of
equipment and programme for sterilization.
Failed or atypical sterilization cycles must be formally investigated.
The sterilization guides from the actual annex are than repeated,
with a little extra information:
• Heat sterilization is still the method of choice
• When revalidation of the process is necessary, is explained
• Next to autoclave tape, irradiation indicator is added
• The part about biological indicators is more extended, without
actually telling us a lot more
The rest of the sterilization part is new, and again, sometimes strict:
• “In house” sterilized material not used immediately after
sterilization should be stored, using sealed packaging, in a grade
B environment
If packed with multiple sterile packaging layers (multiple is at
least 2?), need not to be stored in grade B.
Justification is needed, e.g. multiple sterile coverings that can be
removed at each transfer from lower to higher grade (answer to
the question above)
• Transfer of above sterilized sealed packaged material to A/B area
should be done using validated methods with accompanying
disinfection of the exterior of the sealed packaging. Packaging
may be multi-layered to allow removal of a single layer at each
interface to a higher grade (is this not a contradiction to the
point above?)
• Transfer of materials, equipment and components into an
aseptic processing area should be via an unidirectional process,
examples are given
• When using sealed packaging or containers for sterilization, the
integrity of the sterile protective banner should be qualified for
the maximum hold time, and inspection of each sterile item
prior to use is needed
• For materials that cannot be sterilized, validated disinfection and
transfer is needed.
These items should be included in the environmental
monitoring program
• Validation of a depyrogenation process should demonstrate in
a minimum 3 log reduction in endotoxin. There is no additional
requirement to demonstrate sterilization in these cases.
16
Sterilization by heat
The current guideline is kept, with a few minor extra explanations.
At the beginning of this part, the difference between moist and dry
heat and where they should be used is thoroughly described.
The following is also added, but I don’t see why this is added in the
‘sterilization-by-heat’-part:
“In those cases where parametric release has been authorized, a
robust system should be applied to the product lifecycle validation
and the routine monitoring of the manufacturing process. This
system should be periodically reviewed.”
Moist heat sterilization
Some parts of the current guideline are kept, but moistly, this part is
new, and much more detailed. (see also Steam used for Sterilization
in the utilities chapter)
Next to temperature and pressure, also time should be used to
monitor the process.
Each item sterilized should be inspected for damage, seal and
packaging integrity, both on removal from the autoclave and prior
to use.
Moisture should also be checked after autoclaving, load dryness
should be confirmed as a part of sterilization process acceptance.
SIP (steam-in-place) was not described before, but it is now:
SIP systems should be appropriately designed and validated.
Monitoring of temperature, pressure and time should be done at
critical locations. These critical locations should be demonstrated
as being representative, and correlated with, the slowest to heat
locations during initial and routine validation. it may be necessary to
record the temperature at condensate drain locations throughout
the sterilization period.
Once a system has been sterilized by SIP it should remain integral
prior to use, the maximum duration of the hold time should be
qualified.
Critical validation parameters are equilibration time, exposure time,
correlation of pressure and temperature and maximum temperature
range during exposure for porous cycles and temperature, time and
Fo for fluid cycles.
If a vacuum phase is part of the cycle, frequent leak tests are
necessary.
If air purging is part of the sterilization process, assurance of air
removal is needed, loads should have free draining to prevent the
build-up of condensate.
Damage of containers produced by Blow-Fill-Seal and Form-Fill-
Seal should be prevented by setting correct counter pressure and
loading patterns.
Care should be taken to ensure that materials or equipment are
not contaminated after the sterilization exposure phase of the
cycle due to the introduction of non-sterile air into the chamber
during subsequent phases; typically only sterile filtered air would be
introduced into the chamber during these phases.
Dry heat sterilization
The current guideline only speaks about air circulation, HEPA
filtering of the air, and the use of endotoxins of as part of the
validation. The new annex is, again, much more comprehensive.
Combination of time and temperature exposure should produce
an adequate and reproducible level of lethality and/or pyrogen
(endotoxin) inactivation/removal.
Within depyrogenation tunnels (see current guideline), airflow
patterns should protect the integrity and performance of the
sterilizing zone. All air should pass a HEPA filter and filter integrity
testing is needed. Any tunnel parts that come into contact with
sterilized components should be appropriately sterilized or
disinfected.
Critical process parameters are:
a) Belt speed or dwell time within sterilizing zone.
b) Temperature – Minimum and maximum temperatures.
c) Heat penetration of material/article.
d) Heat distribution/uniformity.
e) Airflows – correlated with the heat distribution and penetration
studies.
When using endotoxin spiked containers (during validation?), full
reconciliation must be performed.
Dry heat ovens should be maintained at a positive pressure to lower
grade areas. All air entering the oven should pass through a HEPA
filter.
Critical process parameters are:
a) Temperature.
b) Exposure period/time.
c) Chamber pressure.
d) Heat penetration of material/article (slow to heat spots and
different loads).
e) Heat distribution/uniformity
Sterilization by radiation
This part is (the only?) part with less information in the new annex
compared with the current. This is because in the meantime,
Annex 12 (Use of Ionising Radiation in the Manufacture of Medicinal
Products) was written, which is referenced in the new annex.
Sterilization with ethylene oxide
Almost exactly the same (current and new annex), with one major
difference:
Both the current and new annex describe some critical process
parameters (gas concentration, temperature and pressure; exposure
time; relative humidity; …)
The current annex says this information must be part of the batch
record. This is not repeated in the new annex, here it is said that
these parameters must be part of the validation and routine
monitoring. More explanation is given about the design of the
filtration system, but again, all rather logical.
17
Filtration of medicinal products which cannot be
sterilized in their final container
Both the current and new annex begin with explaining the risks,
only if nothing else is possible, filtration should be used as the sole
sterilizing process.
A lot is new, but logical.
The selection of the filtration system should be based on CQA’s of
the products, and should not alter the quality of the product.
Adsorption and extraction/leaching should be evaluated.
Like in most chapters of the new annex, the parameters that should
be considered in validation are given:
a) If the system is flushed or integrity tested in-situ with a fluid
other than the product, then flushing with the product should
be part of the process.
b) The wetting fluid used for filter integrity testing based on filter
manufacturer’s recommendation or the fluid to be filtered.
For the latter, the appropriate integrity test value specification
should be established.
c) Filtration process conditions including:
• Fluid prefiltration holding time and effect on bioburden.
• Filter conditioning, with fluid if necessary.
• Maximum filtration time/total time filter is in contact with
fluid.
• Flow rate.
• Filtration volume.
• Temperature.
• The time taken to filter a known volume of bulk solution
and the pressure difference to be used across the filter.
Any significant differences from those validated to those
observed during routine manufacturing should be noted
and investigated. Results of these checks should be
included in the batch record.
Other novelties:
• Wetting of the filter must be avoided for gas filtration,
hydrophobic filters are recommended
• All sterilizing-grade filters should pass integrity testing where
serial filtration is a process requirement
• If a redundant sterilizing filter is used, the additional filter does
not require post-integrity testing unless the primary sterilizing
filter fails
Filter integrity testing is the same, with two additions: integrity of the
filter should be verified by testing before use, in case of damage and
loss of integrity caused by processing, and after use. Not sure what is
meant with this first addition, is there a difference with the after use
(assuming you stop production when you see damaging has taken
place)?
Second addition is that for small batch sizes, filter integrity testing
may not be possible, alternatives can be used, as long as formal risk
assessments are done.
Form-Fill-Seal technology
This part is new.
A short explanation is given about FFS (Form-Fill-Seal), BFS (Blow-
Fill-Seal) and VFFS (Vertical-Form-Fill-Seal).
100 % integrity testing for VFFS is needed.
Seal integrity critical parameters for validation and control must
include:
• Seal strength (should be monitored routinely)
• Seal uniformity (should be monitored routinely)
• Sealing temperatures and pressures
• Sealing times
• Dwell time for sealing
Samples of filled containers should be tested for ease-of-opening
and uniformity.
Blow-Fill-Seal technology
The content of the current annex is kept, with quite some extra’s.
Also, some current guidances are only applicable for Shuttle-type,
others also for rotary-type equipment.
The aspects to consider in the risk management of the machine’s
design and operational controls are more extended:
a) Determination of the “critical zone” that should be protected
from contamination, and its control.
b) Environmental control and monitoring, both of the BFS
machine and the background in which it is placed.
c) Integrity testing of the BFS product pathways.
d) Duration of the batch or filling campaign.
e) Control of polymer starting material.
f) Cleaning-in-place and sterilization-in-place of equipment, and
air and product pathways.
Environmental control and monitoring should take generated gas
flow paths and high heat outputs of the process into consideration.
The area between parison cutting and mould sealing (Shuttle-type)
should be covered by a flow of air to provide grade A.
External contamination of the polymer should be prevented.
The aseptic filling procedure must be well described and simulated,
also for interventions.
Critical operating parameters that should be taken into
consideration during process validation are filling speed, extrusion
temperature and filling times.
Samples of filled containers should be tested for ease-of opening
and wall thickness.
Lyophilization
New, but the content is logic:
• Sterility must be maintained until completion of lyophilization
process, equipment must be sterilized before use,
contamination must be prevented
• Maximum permitted leakage of air into the lyophilizer should be
specified, integrity of the system should be monitored
• Loading patterns must be documented
• Transport to the lyophilizer and loading should take place under
grade A environment, unsealed containers (or if seating of the
stoppers is not completed before opening) must be kept under
grade A environment
• All utensils used during transfer, loading and unloading should
be subjected to a validated sterilization process.
18
Closed systems
New.
Closed systems can be single use (see Single use systems) or fixed.
No groundbreaking novelties here:
• Sterility within the system must be maintained
• Tubing that is not assembled prior to sterilization, should be
connected aseptically
• Product contact surfaces must be sterile
• Integrity of the components must be assured, and the
background will rely on this:
o If there is a high risk that the system will not remain integral
during processing (is this a closed system?), it should be
located in a grade A
o If the system remains integral at every usage, grade D can
be considered
• System integrity tests should be in place
SIP (steam-in-place) was not described before,
but it is now:
SIP systems should be appropriately designed and
validated. Monitoring of temperature, pressure
and time should be done at critical locations. These
critical locations should be demonstrated as being
representative, and correlated with, the slowest to
heat locations during initial and routine validation.
it may be necessary to record the temperature
at condensate drain locations throughout the
sterilization period.
Once a system has been sterilized by SIP it should
remain integral prior to use, the maximum duration
of the hold time should be qualified.
Single use systems
Also new.
A definition is given (Single use systems (SUS) are designed to
replace reusable equipment!)
Specific risks associated with SUS are:
a) Interaction between the product and product contact surface
(adsorption, leachable and extractables).
b) More fragile than fixed reusable systems.
c) Increase in number and complexity of manual operations and
connections made.
d) Design of the assembly.
e) Performance of the pre-use integrity testing for sterilizing grade
filters. (Refer to clause 8.84.)
f) Integrity testing.
g) Pin-hole and leakage.
h) The potential for compromising the system at the point of
opening the outer packaging.
i) Assessment of suppliers of disposable systems (including
sterilization of these disposable systems.
j) Risk of particulate contamination.
The rest of this part deals with extractables and leachables, system
integrity and critical manual handling of the system.
SUS components must have similar receipt and inspection
procedures as packaging materials.
19
9. Viable and non-viable environment &
process monitoring
Current Annex: Clean room and clean air device monitoring.
This is a much shorter part, which tells us to routinely monitor clean rooms. Grade A
zones should be continuously monitored, except if the contaminants during process
possibly damage the particle counter. The same is recommended for grade B zones.
See also 5. Premises
In the new Annex 1, room classification and qualification is
given in chapter 5. Premises, while in this chapter, monitoring of
environment & process is described. In the current annex, the
chapter “Clean room and clean air device monitoring” describes
both. For the main differences between current and new annex, see
5. Premises.
Environmental monitoring
The environmental monitoring must be based on appropriate risk
assessments, which should be re-evaluated at defined intervals
(note the difference of this term compared to regularly) in order to
confirm the effectiveness of the site’s environmental monitoring
program.
Monitoring should be considered (by risk assessments of course) in
operation, throughout all stages, including equipment set up, and
outside of operations, e.g. pre disinfection, post disinfection, prior to
start of manufacturing and after a shutdown period.
Sampling of grade A should be performed at locations posing the
highest risk, sounds logical, and, because of all the emphasis on risk
assessments, pointless to add.
Alert and action limits should be set for particulate and
microbiological monitoring, with alert limits based on PQ tests or
trends and subjected to periodic review. Some well-known guidance
regarding the meaning of alert and action limits are given.
Repeated here (and thus important): Surfaces and personnel should
be monitored after critical operations. Results from monitoring
should be considered when reviewing batch documentation for
finished product release.
Non-viable monitoring
Recommended limits are given, both for particles ≥ 0.5 and 5 µm.
The limits are the same as in the current annex, with one difference
for grade D in operation. Limits were “not defined” in the current,
and “based on the risk assessment” in the new annex.
Note the difference (new) for qualification and monitoring: for
qualification, only particles ≥ 0.5 µm are in scope, for monitoring,
also particles ≥ 5 µm must me measured.
Explanation why is given: particles ≥ 5 µm are an important
diagnostic for detection of machine, equipment and HVAC failure.
Occasional indication of ≥ 5 µm particles may be considered false
counts due to electronic noise, stray light or coincidence, but
regular counting of low levels may be indicative of a possible
contamination. More explanations of the difference between
occasional and regular is not given, so, exceptional in this annex,
room for interpretation is present.
In grade A zones, monitoring must be done for the full duration of
processing, including assembly. At least a sample size of 28 litres per
minute should me monitored.
A similar system is recommended for B zones, sample frequency
may be decreased. Everything based, again, on risk management,
especially regarding product sterility assurance.
C and D areas should be monitored in accordance with QRM
principles, effective trend analysis must be made possible.
Some explanations are given what to do when contaminants are
present that could damage the particle counter (classify before and
after, and during simulated operations) and when powdery products
are manufactured (also, monitor before and after operations)
Viable monitoring
Should be used where aseptic operations are performed, using
settle plates, volumetric air, glove print and surface sampling, and
also sampling of personnel.
Again, during full operations, including set up, monitoring in A en B
areas must be done.
20
Some logical guidance is given:
• Rapid microbial monitoring may be used, off course when
validated and proven equivalent to established methodology.
• Sampling methods should not pose a contamination risk to
operations
• E.g. after validation, cleaning and inspection, microbiological
monitoring should also be performed
• Trending must be used (and possible ways of trending are
explained
• Detected micro-organisms in A and B zone (consider to do
the same in C and D) should be identified, impact on product
quality must be evaluated
Recommended maximum limits for microbial contamination are
the same as in the current Annex, with one difference: the less than
1 cfu/m³;plate;glove in grade A now became 1, with the note that
0 cfu should be recovered, starting from 1, an investigation should
be started.
Aseptic process simulation (APS)
APS is the term used for what is known in industry as media fill. In the
current annex, the terms media fill and process simulation are used
a few times, now they only speak about APS, just one time, the term
media fill is used in the new annex.
The part on APS, or media fill, if you want, has significantly grown,
from less than a page to more than 3 pages.
For APS, a sterile nutrient media and/or placebo, with the ability to
imitate product characteristics, should be used.
The process simulation test should imitate as closely as possible the
routine process, and include all the critical manufacturing steps. An
enumeration of what this means, and what should be included is
given.
The same manipulations and interventions both during normal
production as in worst-case situations, must be taken into account.
A list of allowed interventions should be made, both inherent and
corrective.
Developing the process simulation test plan, risk management
principles should be used, considering:
• Identification of worst case conditions covering the relevant
variables and their microbiological impact
• Determining the representative sizes of container/closure
combinations for validation, bracketing or matrix approach may
be used
• Volume filled per container: to make sure media contacts
all equipment and component surfaces that may cause
contamination
• Holding times
• Ensuring that any contamination is detectable
• requirement for substitution of any inert gas used in the
routine aseptic manufacturing process by air, unless anaerobic
simulation is intended
• duration of the process simulation must ensure it is conducted
over the maximum permitted filling time
• Simulating normal aseptic manufacturing interruptions where
the process is idle
• Special requirements and considerations for manually intensive
operations
• Beginning, end and duration in the case of campaign
manufacturing
• Specific risks and aspects of technologies such as RABS,
isolators, BFS, …)
APS should be performed as initial validation, generally with 3
consecutive simulation tests per shift, and after any significant
modification.
Periodic revalidation (APS) should be repeated:
• Every six months for each process and filling line
• Annually for each operator, (restrict entry of an operator when
results indicate a failed qualification)
• Before shut down, long periods of inactivity or before
decommissioning or relocation of a line: this means you don’t
only use APS as some kind of validation, but also as a kind of “as
found” calibration, too make sure you were working aseptically
in the past period: see also the guidance given a page later:
after a failed APS, review of all appropriate records since last
successful APS must be done.
For manual filling, each product, container closure, equipment train
and operator should be revalidated every 6 months. Repeating the
initial validation should be done when:
• Revalidation has failed (and consecutive corrective actions have
been taken)
• Long period of not operating the process
• Change to process, equipment, personnel procedures or
environment
Just like in the current annex, some guidance is given about the
number units filled for APS. For batches under 5000 units, media fills
(only place this term is used, sentence is exact copy of the one in the
current annex) should at least be the size of ...the production batch.
Target should be zero growth, any contaminated unit should result
in an investigation and CAPA. Very clear: effectiveness check of
the CAPA should be done with a new APS, typically, 3 successful
consecutive repeat APS would be expected. (In the current annex,
revalidation should be done, for batches more than 5000 units,
starting from 2 contaminated units).
Another very specific guidance: Before incubation, filled APS units
must be agitated, swirled or inverted to ensure contact of the
media with all interior surfaces. Incubation should be done in clear
containers to ensure visual detection of growth.
Risk based manufacturing on a line following APS is allowed, but
all products should be quarantined until resolution of the APS has
occurred.
All APS runs should be fully documented, including a reconciliation
of units processed.
APS (Aseptic Process Simulation) is the term used for
what is known in industry as media fill. In the current
annex, the terms media fill and process simulation are
used a few times, now they only speak about APS.
Just one time, the term media fill is used in the new
annex.
21
10. Quality Control (QC)
In the current annex, there is told that:
• Sterility test of finished product is only one (the last) in a series
of control measures (repeated in the new annex)
• Special attention towards validation and monitoring of the
process in case of parametric release (repeated)
• Samples taken should be representative of the whole batch,
but should in particular include samples most at risk of
contamination:
o Beginning, end and after interventions (repeated)
o Coolest part in case of heat sterilization (repeated)
o Each sterilized load should be considered as a different
batch (new)
o Products in different lyophilization loads (new)
Everything else is new:
• Microbiological contamination of starting materials should be
minimal
• Bioburden assay should be performed for both aseptically
filled and terminally sterilized products, results are part of the
final batch review. Contamination limits immediately before
sterilization are needed, because they are related to the
efficiency of the method.
• Where overkill sterilization parameters are set for terminally
sterilized products, bioburden should be monitored at suitable
scheduled intervals
• The sterility test should be performed under at least the same
aseptic conditions as the manufacture of the products
• Decontaminating the sterility samples prior to testing should
not negatively impact the test method
• Media used for APS should be tested for growth promotion
capability
• Environmental monitoring in A and B areas should be part of
product batch release
• Rapid microbial methods can be considered, off course when
validated.
22
11 Glossary

The new annex ends with a 5 page long glossary with definitions of all important terminology used.

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