GMP Requirements for Sterile Products Manufacturing

Annex 6. TRS 961, 2011
GMP Requirements for Sterile
Products manufacturing
Module-1
Prepared by: Surafel
Kebede(Bpharm,MBA)
safokebede@gmail.com
2

Table of contents
1. Introduction
2. General Considerations
3. Quality control
4. Sanitation
5. Manufacture of sterile preparations
6. Sterilization
7. Isolator technology
8. Blow/ Fill/ Seal Technology
9. Personnel
10. Premises
11. Equipment
12. Finishing of sterile products
3

Objectives
 To review basic GMP requirements in the manufacture of
sterile pharmaceutical products
 To review air classifications for activities related to the
manufacture of sterile products
 To review quality assurance aspects in the manufacture and
control of sterile products
4

1.Introduction
This training module is prepared based on (Annex 6. TRS 961, 2011) &
trainees are highly recommended to read this document together with
Annex 6. TRS 961, 2011.
Note that the italicized sentences are taken directly from the guideline
(Annex 6. TRS 961, 2011) and further explained in detail.
These requirements(Annex 6. TRS 961, 2011) are additional rather than
replacement and emphasize specific points for the manufacture of sterile
preparations to minimize the risks of:
 Microbiological contamination
-Viruses, bacteria, fungus, etc
 Particulate Matter contamination
-Dust, fibers, or other materials
 Pyrogen contamination
-Fever inducing substance, could be chemicals or Endotoxin
5

 The production of sterile preparations should be carried out in
clean areas(cleanrooms).
Cleanroom(clean zone): is a room in which the concentration of
airborne particles is controlled, and which is constructed and used
in a manner to minimize the introduction, generation, and
retention of particles inside the room and in which other relevant
parameters, e.g. temperature, humidity, and pressure, are
controlled as necessary.(ISO standard 14644-1)
6

Clean area: An area with defined environmental control of
particulate and microbial contamination, constructed and used in
such a way as to reduce the introduction, generation, and
retention of contaminants within the area(GMP and inspection,
WHO).
 Clean areas(cleanrooms) should be maintained to an
appropriate standard of cleanliness and supplied with air
that has passed through filters of the required efficiency.
7

Air Filters used in Cleanrooms
 The air supplied to a cleanroom must be filtered to ensure the
removal of particles and micro-organisms.
 High Efficiency Particulate Air (HEPA) filters filtered
cleanroom air, are the most efficient air filters available.
 (HEPA) filter can remove up to 99.995% o f particles with a
size of 0.3 µm or larger.
 Currently better filters are used and these are known as Ultra
Low Penetration Air (ULPA) filters.
 An ULPA filter will have an efficiency greater than 99.999%
against 0.1-0.2 µm particles. These ULPA filters are constructed
and function in the same way as a HEPA filter.
General...
8

It is generally accepted that:
 For cleanrooms of IS0 Class 6 (Class 1,000) and poorer quality,
HEPA filters are used with turbulent ventilation to meet the
cleanroom classification.
 For IS0 Class 5 (Class l00), HEPA filters that completely cover
the ceiling are used to supply unidirectional flow down through
the cleanroom.
 For IS0 Class 4 (Class 10) or lower, ULPA filters should be
used with unidirectional flow.
General...
HEPA filters
9

Filter classification
General...
Classification of filters according to the EN 1822
10

Filter classification
General...
Classification of High Efficiency Filters EPA and HEPA according EN 1822:2009
11

 High-efficiency particulate air (HEPA) filters should be
subjected to an installed filter leakage test in accordance with
ISO 14644-3 (3) at a recommended interval of every 6 months,
but not exceeding 12 months. The purpose of performing regular
leak tests is to ensure the filter media, filter frame and filter seal
are free from leaks. The aerosol selected for HEPA leak testing
should not support microbial growth and should be composed of
a sufficient number or mass of particles. HEPA filter patching is
allowed at the filter manufacturer and in situ operation provided
that the patch sizes and procedures follow the recommendations
of ISO 1822-4 (4).
General...
12

General...
Deep-pleated HEPA filter
13

HEPA filter leakage test
 Testing a high efficiency filter and its filter housing for leaks is
normally done with an artificial test aerosol. This is generated
by smoke generator and injected into the ductwork system so
that there is a suitable concentration behind the high efficiency
filter. Any problems are found by scanning the filter system for
test particles that leak through.
 High efficiency filters should have been supplied with the
correct particle removal efficiency. A secondary function of the
installation leak test is to check that the correct efficiency of
filter has been supplied.
General...
14

 This USA test originally used thermally generated particles of
di-octyl phthalate (DOP) with an average size of 0.3 µm to test
the efficiency of HEPA filters. However, other oils such as
poly-alpha olefin (PAO) or di-octyl sebacate (DOS) have
replaced DOP(because it is carcinogenic). Heating such an oil
produces an oil mist, and the efficiency against this challenge is
determined.
 DOP aerosol generator is placed upstream of HEPA filter to
introduce challenge particles (0.3µm aerosol of DOP ) and
Scanning with a photometer is done downstream to detect
leaks (See Video).
General...
15

Acceptance criteria
 For HEPA filters(eg. H 14 type) of 99.995% efficiency, the
challenge aerosol penetration should be lower or equal to
0.005% of the upstream concentration.
General...
Scanning HEPA filter with photometer
DOP aerosol generator
16

Is HEPA filter patching recommended?
 EN 1822-4, Section 8.5.4 states: “A filter may be repaired if
necessary and shall then be retested” and further notes: “All
repairs together (including those made by the filter
manufacturer) shall not block or restrict more than 0.5% of the
filter face area (not including the frame) and the maximum
length of each single repair shall not exceed 3.0 cm. Alternative
repair criteria may be otherwise agreed between buyer and
seller.”
General...
17

Types of Cleanrooms
 Cleanrooms have evolved into two major types and they are
differentiated by their method of ventilation.
 These are turbulently ventilated and unidirectional flow
cleanrooms.
a. Turbulently ventilated
 Turbulently ventilated cleanrooms are also known as ‘non-
unidirectional’.
 A turbulently ventilated room receiving clean filtered air
through air diffusers in the ceiling. This air mixes with the room
air and removes airborne contamination through air extracts at
the bottom(usually) of the walls.
General...
18

 In this style of cleanroom, the contamination generated by people
and machinery is mixed and diluted with the supply air and then
removed.
b. Unidirectional flow
 Unidirectional flow cleanrooms (either vertical or horizontal type)
were originally known as ‘laminar flow (LAF)’ cleanrooms.
 High efficiency filters are installed across a whole ceiling (or wall
in some systems) and these supply air. This air sweeps across the
room in a unidirectional way at a speed of around 0.45m/s (90
ft/min) and exits through the floor, thus removing the airborne
contamination from the room.
 The unidirectional type of cleanroom uses very much more air than
the turbulently ventilated type, and gives superior cleanliness.
General...
19

 The air supplied to unidirectional flow rooms is many times
greater (10 or 100 of times) than that supplied to a turbulently
ventilated room. These cleanrooms are therefore very much
more expensive to build and run.
 In the case of turbulently ventilated cleanrooms the air supply
and extract volumes should be measured. In the case of
unidirectional airflow it should be the air velocity.
General...
Flow measuring hood Digital Anemometer 20

General...
Turbulently ventilated type of cleanroom Unidirectional flow type of cleanroom
21

General...
 Entry to cleanrooms should be through airlocks for personnel
and/or for equipment and materials.
Airlock: An enclosed space with two or more doors, which is
interposed between two or more rooms, e.g. of differing classes of
cleanliness, for the purpose of controlling the airflow between
those rooms when they need to be entered. An airlock is designed
for use either by people or for goods and/or equipment. (GMP and
inspection, WHO).
 Airlocks are called PAL (Personnel Air Lock) when used for
personnel and MAL (Material Air Lock) when used for
transferring the material.
22

Material and personnel air locks
23

Types of airlocks
 In general, there are three basic airlock design that can be
combined or used individually to protect the clean room and/or
prevent cross contamination between two adjacent areas of
different process operations.
1.Cascade Airlock
High pressure on one side of the airlock and low pressure on
the other side.
The cascading pressure airlock is used to protect clean areas from
adjacent areas with lower required cleanliness.
24

Types of...
APPLICATION
 Any manufacturing facilities where the product requires
protection from particulate but the people outside the cleanroom
do not need protection from the product in the cleanrooms.
 It can be used in tablet manufacturing facilities where higher
pressure is in corridor than production area to keep the drug
inside production area.
 What about HUMAN WELL PHARMA????
25

2. Bubble Airlock
 High pressure inside the airlock and low pressure on both outer
sides.
 Because it runs at positive pressure to both areas, it creates a
barrier where contaminants within either area are pushed back
into their own respective areas.
 The air being used to pressurize the bubble needs to be of higher
quality than both the internal and external zone.
Application
 Is commonly used in injectables manufacturing facilities so that
air contaminants from outside may not contaminate drug
substance.
 Used in, areas where the products needs protection and the
people external to the cleanrooms require protection from the
product.
27

3. Sink Airlock
 low pressure inside the airlock and high pressure on both outer
sides.
 A sink airlock is one where it runs a negative pressure in
relation to the internal and external zone.
Application
 In many research facilities, substances that are experimented on
are highly dangerous, and it is essential to keep them from being
exposed. During a few types of production processes in a
cleanroom, air from a contaminated area has to be contained in
one place.
29

General...
 The various operations of component preparation (containers and
closures), product preparation, filling and sterilization should be
carried out in separate areas within a clean area. These clean
areas are classified into four grades (A,B,C,D).
 Component: is any ingredient intended for use in the
manufacture of a drug product, including those that may not
appear in the final drug product. (CFR Part 210, FDA)
 Component: is any raw material, substance, piece, part, software,
firmware, labeling, or assembly which is intended to be included
as part of the finished, packaged, and labeled (medical) device.
(CFR Part 820, FDA)
31

General...
 Component preparation room shall be of Grade C or D air quality
and shall be entered by personnel via a personnel changing room
of a similar grade.
 Manufacturing operations of sterile products are divided into two
categories: first, those where the product is terminally sterilized,
and second, those which are conducted aseptically at some or all
stages.
32

General ...
Cleanrooms layout
33

3. Quality control
Parenteral Quality Control Tests of finished products.
The main tests are:
1.Sterility testing
2.Pyrogen/endotoxin testing
3.Particulate matter testing (visible and sub-visible)
4.Package integrity testing
5.Content uniformity
6.Uniformity of weight/volume
34

Quality…
 The sterility test applied to the finished product should only be
regarded as the last in a series of control measures by which
sterility is assured. The test should be validated for the
product(s) concerned.
 Samples taken for sterility testing should be representative of
the whole of the batch, but should, in particular, include
samples taken from parts of the batch considered to be most at
risk of contamination, for example:
(a) for products that have been filled aseptically, samples
should include containers filled at the beginning and end of the
batch and after any significant interruption of work;
(b) for products that have been heat sterilized in their final
containers, consideration should be given to taking samples
from that part of the load that is potentially the coolest. 35

Sample size for sterility test (USP36-NF31)
36

Quality…
 The sterility of the finished product is assured by validation of
the sterilization cycle in the case of terminally sterilized
products, and by “media simulation” or “media fill” runs for
aseptically processed products.
 A “media fill” (sometimes known as a “process simulation”) is the
performance of an aseptic manufacturing procedure using a sterile
microbiological growth medium in place of the drug solution.
Microbiological growth medium is used in place of the drug solution
during media fills to test whether the aseptic procedures are adequate to
prevent contamination during actual drug production. A media fill is one
part of the validation of an aseptic manufacturing process.
 Batch-processing records and, in the case of aseptic processing,
environmental quality records, should be examined in
conjunction with the results of the sterility tests.
39

Quality…
 The sterility test procedure should be validated for a given
product. Pharmacopoeial methods should be used for the
validation and performance of the sterility test. In those cases
where parametric release has been authorized in place of
sterility testing special attention should be paid to the
validation and the monitoring of the entire manufacturing
process.
Parametric release
 A system of release that gives the assurance that product is of
the intended quality based on information collected during the
manufacturing process and on the compliance with specific
GMP requirements related to Parametric Release (EU Annex
17, 2001).
40

Quality…
 At present Parametric release can only be approved for
products terminally sterilized in their final container.
Parametric release, in lieu of end product sterility testing, is
acceptable when all of the following parameters are met and
documented.
 The sterilization process cycle has been validated to achieve
microbial bioburden reduction to 1 with a minimum safety
factor of an additional six logarithm reduction.
 Cycle validation includes sterilizer heat distribution studies,
heat distribution studies for each load configuration, heat
penetration studies of the product, bioburden studies, and a
lethality study referencing a test organism of known resistance
to the sterilization process.
41

Quality…
 All cycle parameters must be identified by the manufacturer as
critical (e.g., time, temperature, pressure) or non-critical (e.g.,
cooling time, heat-up time).
 Under parametric release, failure of more than one critical
parameter must result in automatic rejection of the sterilizer
load.
 Integrity for each container/closure system has been validated
to prevent in-process and post-process contamination over the
product’s intended shelf life. Validation should include
chemical or microbial ingress tests utilizing units from typical
products.
42

Quality…
 Bioburden testing (covering total aerobic and total spore
counts) is conducted on each batch of pre-sterilized drug
product. Resistance of any spore-forming organism found must
be compared to that of the organism used to validate the
sterilization cycle. The batch is deemed non-sterile if the
bioburden organism is more resistant than the one used in
validation.
 Chemical or biological indicators are included in each truck,
tray, or pallet of each sterilizer load. For chemical indicators,
time/temperature response characteristics and stability are
documented and for each sterilization cycle minimum
degradation values are established. Chemical indicators cannot
be used to evaluate cycle lethality.
43

Quality…
 For injectable products, the water for injection(It is an
intermediate bulk product) and the intermediate and finished
products should be monitored for endotoxins, using an established
pharmacopoeial method that has been validated for each type of
product.
 Intermediate Product: A partly processed material, which
should undergo further preparation steps before it becomes a bulk
product. (EU GMP Guide). Eg bulk solution, granules etc
 Bulk Product: Any product that has completed all processing
stages up to, but not including, final packaging. (GMP and
inspection, WHO). Eg unpacked tablets, sterilized bags etc
44

Quality…
 For large-volume infusion solutions, such monitoring of water or
intermediates should always be done, in addition to any tests
required by an approved monograph for the finished product.
When a sample fails a test, the cause of such failure should be
investigated and remedial action taken where necessary.
Alternative methods to those in the pharmacopoeias may be used
if they are validated, justified and authorized.
45

Quality…
 The use of rapid microbiological methods to replace the
traditional microbiological methods, and to obtain earlier results
on the microbiological quality of, for example, water, the
environment or bioburden, could be considered if appropriately
validated and if a comparative assessment of the proposed rapid
method is performed against the pharmacopoeial method.
 Rapid microbial methods (RMMs): also known as alternative
microbiological methods, are the technologies that allow the user
to get microbiology test results faster compared with traditional
culture-plate methods.
46

Quality…
<1225> VALIDATION OF COMPENDIAL PROCEDURES
‘’Test procedures for assessment of the quality levels of pharmaceutical articles
are subject to various requirements. According to Section 501 of the Federal
Food, Drug, and Cosmetic Act, assays and specifications in monographs of
the United States Pharmacopeia and the National Formulary constitute legal
standards. The Current Good Manufacturing Practice regulations [21 CFR
211.194(a)] require that test methods, which are used for assessing
compliance of pharmaceutical articles with established specifications, must
meet proper standards of accuracy and reliability. Also, according to these
regulations [21 CFR 211.194(a)(2)], users of analytical methods described in
USP–NF are not required to validate the accuracy and reliability of these
methods, but merely verify their suitability under actual conditions of use’’.
(USP36-NF31,2013)
47

4. Sanitation
Sanitation: is the process of keeping places clean and healthy.
Cleaning, Sanitizing, and sterilization
 Cleaning is a process that removes soil and other particulates from
a surface, there by preventing the accumulation of contaminants.
 Sanitizing is a cleaning process that destroys disease-causing
organisms present on surfaces. It reduces the amount of microbial
life on a particular product/item.
 Sanitizing= cleaning + disinfection
 Sterilization is the complete destruction of all microorganisms
including the most resistant bacteria and spores.
48

Sanitation…
 The sanitation of clean areas is particularly important. They
should be cleaned frequently and thoroughly in accordance with
an approved written programme (a programme is a planned series
of future events or performances).
 Monitoring (environmental )should be regularly undertaken in
order to detect the emergence of resistant strains of
microorganisms. Where disinfectants are used, more than one type
should be employed (i.e. disinfectant rotation is important to
reduce resistance strain).
49

Sanitation…
 Monitoring should be regularly undertaken to detect
contamination or the presence of an organism against which the
cleaning procedure is ineffective.
 Interactions between different cleaning materials should be
validated. Appropriate cleaning validation should be carried out
to ensure disinfectant residuals can be detected and are removed
by the cleaning process.
50

Sanitation…
 Disinfectants and detergents should be monitored for
microbiological contamination; dilutions should be kept in
previously cleaned containers and should only be stored for
defined periods unless sterilized.
 Disinfectants and detergents used in grade A and B areas should
be sterilized before use.
 A disinfectant programme should also include a sporicidal agent
since many common disinfectants are ineffective against spores.
 The effectiveness of cleaning and disinfectant procedures should
be demonstrated.
 Fumigation of clean areas may be useful for reducing microbial
contamination in inaccessible places.
51

Sanitation…
Fumigation vs fogging
Fumigation
 Formaldehyde fumigation is effective against all the
microorganisms causing potential problems to pharmaceutical
products.
 In formaldehyde fumigation, formaldehyde is mixed with
potassium permanganate to generate fumes (Formaldehyde vapor).
 Prior to fumigation area should be sealed and air handling units
(HVAC) should be turned off. However , Fumigation with
Formaldehyde is Banned in most of countries as the fumes
generated have been declared Carcinogenic by regulatory
authorities across the globe. And most pharmaceuticals moved
their way towards Fogging.
53

Sanitation…
 After fumigation the formaldehyde should be neutralized with
ammonia. Urotropine (Hexamethylenetetramine) is obtained from
the reaction.
 Hexamethylenetetramine or methenamine is a heterocyclic organic
compound with the formula (CH2)6N4. This white crystalline
compound is highly soluble in water and polar organic solvents.
 Robust cleaning procedure is very mandatory after neutralization.
54

Sanitation…
Fogging
 In Fogging equipment called Fogger is used to
achieve disinfection.
 After formaldehyde the regulatory authorities suggested use
of Hydrogen peroxide for disinfection.
 Hydrogen peroxide is relatively safer disinfectant when compared
with Formaldehyde.
 The mechanism of action of hydrogen peroxide involves chemical
oxidation of cellular components (see video).
55

Sanitation…
 The Fogger is used to generate 5-10 microns particles of
Hydrogen peroxide in Fogger machine, solution of 2% Hydrogen
peroxide with water is placed when Fogger is turned on it will
heat the solution to create fog which is then mixed with jet of air
which will atomize the fog into particles ranging from 5-10
microns and disperse them in air.
 After fogging cleaning is not required as Hydrogen peroxide
automatically decomposes to water and oxygen after Fogging.
 Time of the fumigation or fogging can be validated for better
results of fumigation.
 Area should be free from fumigating chemicals properly before
use.
56

Sanitation…
 In order to control the microbiological cleanliness of the various
grades in operation, the clean areas should be monitored.
 Where aseptic operations are performed, monitoring (microbial)
should be frequent and methods such as settle plates, and
volumetric air and surface sampling (e.g. swabs and contact
plates) should be used.
 The zones should not be contaminated through the sampling
methods used in the operations.
 The results of monitoring should be considered when batch
documentation for release of the finished product is reviewed.
Both surfaces and personnel should be monitored after critical
operations.
57

Sanitation…
Environmental monitoring in cleanroom
 Routine monitoring of airborne particle concentration and other
parameters shall be performed according to a written plan &
procedure.
 The airborne-particle monitoring plan shall be based on risk
assessment related to the application of the installation.
 Environmental monitorings are:
1. Physical monitoring:- particle counting and air pressure
difference monitoring.
2. Microbiological monitoring
58

Sanitation…
1. Physical monitoring
1.1. particle counting
 An instrument known as a ‘particle counter’ is used to count and
size particles in the air of a cleanroom.
Hand-held particle counter Bench-Top Particle Counter
59

Sanitation…
1.2. Air Pressure Difference Monitoring
 It is necessary to ensure that air moves in a cleanroom suite from
a clean to a less-clean area, and not vice-versa.
 The higher quality cleanrooms should therefore have a higher
pressure than adjacent less-clean areas.
 The units of measurement used to register pressure differences are
Pascals, although older units such as inch water gauge are
sometimes used (12Pa = 0.05 inch water gauge).
 A pressure difference of 10 or 15 Pa is generally accepted as that
which should be established between clean areas. 15 Pa is
commonly used between a cleanroom and an unclassified room,
and 10 Pa between two cleanrooms.
60

Sanitation…
 A manometer capable of reading pressure differences in the range
of 0-60 Pa (0-0.25 inch water) is required for measuring the
pressure difference between rooms. This is usually an inclined
manometer, magnehelic gauge, or electronic manometer.
inclined manometer magnehelic manometer electronic manometer
61

2. Microbiological testing
It is common to sample the air and surfaces of the cleanroom, as
well as the personnel working in the cleanroom.
2.1. Microbial sampling of the air
 Several types of apparatus exist for counting micro-organisms
in the air of cleanrooms. These samplers are sometimes known
as ‘volumetric’ air samplers because a given volume of air is
sampled. Many types of samplers have been invented for
sampling micro-organisms in the air.
Sanitation…
Air sampler 62

2.2. Microbial surface sampling
2.2.1. Contact surface
 Contact plates and strips are used when the cleanroom surface to
be sampled is relatively flat. If plates are used, then RODAC
(Replicate Organisms Detection And Counting) dishes of the type
shown in Figure below are used. These dishes are often 55 mm in
diameter with the inner dish covered by a lid resting on a lip.
Pouring 15.5 m1 to 16 ml of agar medium into the central
chamber fills it and gives an agar meniscus that stands proud of
the rim. The agar is rolled over the cleanroom surface to be
sampled. Microorganisms will stick to the agar and when the dish
is incubated for a suitable time and temperature, the micro-
organisms will grow into colonies that can be counted.
Sanitation…
63

 When disinfectants are used there is likely to be a residue left
on the surface that is sampled. The residue may stop the growth
of micro-organisms and chemicals that neutralize the action of
disinfectants should be incorporated into the agar medium to
prevent this.
Sanitation…
Contact plate 64

 Agar contact strips of the type shown in figure below are also
used to sample surfaces. These strips are removed from their
container and applied to the surface to be sampled. The micro-
organisms stick to the agar surface and the number ascertained by
incubation and counting the colonies that grow.
Sanitation…
Contact strips 65

2.2.2. swabbing
 To sample uneven surfaces, a commonly used method is the
application of a bud swab made from a material such as cotton.
At its simplest, a sterile swab is randomly rubbed over the
cleanroom surface to be sampled and then rubbed over an agar
plate. The plate is then incubated and the microbial count
determined. To improve the efficiency and reproducibility, the
swab should be dampened with a sterile liquid such as saline and a
known surface area should be sampled
Sanitation…
swabbing
66

2.3. Personnel sampling
Personnel are the primary source of micro-organisms in a
cleanroom, and it may be necessary to monitor them to ensure
that there is no unusually high dispensers of micro-organisms
working within the cleanroom.
2.3.1. Finger dabs
 The person’s fingers tips, or their gloved hand, is pressed or
wiped on an agar plate and the number of micro-organisms
ascertained.
Sanitation…
Finger dabs
67

3.3.2. Contact plates or strips
The person’s garments are sampled by pressing the plate or strip
onto their clothing. This is best done as they come out of the
cleanroom.
Sanitation…
68

3.3.3. Body box
 Through a HEPA filter in the top of the box, bacteria and particle-
free air is supplied. A volunteer enters the box wearing the
clothing to be studied. After the contamination in the box has been
blown out, the volunteer starts to exercise to the beat of a
metronome. The number of particles and bacteria dispersed per
minute are then counted.
Sanitation…
69

Sanitation…
Body box
70
metronome
bacterial and particle sampler

5. Manufacture of sterile preparations
 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 minimize the risks of particulate or
microbiological contamination of the product or materials
being handled.
71

Manufacture of…
 People are normally the major source of microorganisms
(besides water and equipments) in a cleanroom. Testing for
micro-organisms in the ‘as built’ or the ‘at rest’ operational
conditions will therefore be of little value.
 However, when a cleanroom is fully operational, micro-
organisms are continually dispersed from people in the room. It
will therefore be necessary to monitor the cleanroom to
demonstrate that a pre-determined concentration is not
exceeded.
72

Occupancy states of cleanrooms
The occupancy states defined in IS0 14644-1 are as follows:
1. As built: the condition where the installation is complete
with all services connected and functioning, but with no
production equipment, materials or personnel present.
73

Occupancy…
2. At-rest: The condition where the installation is complete with
equipment installed and operating in a manner agreed between
the customer and supplier, but with no personnel present.
74

Occupancy…
3. In Operation: The condition where the installation is
functioning in the specified manner, with the specified number
of personnel present and working in the manner agreed upon.
75

Manufacture….
Cleanroom classification standards
Historical Background
 It can be argued, with justification, that the first standard
written for cleanrooms was published by the American
Airforce on March 1961 and known as Technical Manual
(T.O.) 00-25-203.
 This considered cleanroom design and airborne particle
standards, as well as operating procedures such as: entry
procedures; clothing; restriction of certain articles; cleaning of
materials; procedures for cleaning the room.
76

Manufacture….
 However, the standard that had the most influence on the design
and operation of cleanrooms, and is the basis of most world
cleanroom standards, including ISO standard 14644-1, was
Federal Standard 209.
 The Sandia Corporation team that invented the unidirectional
concept, aided by others from the USA military, industry and
governmental agencies, produced the first Federal Standard 209
in 1963.
 However, the federal standard was cancelled on November 29,
2001.
77

Manufacture….
FED-STD-209E
“The U.S. General Services Administration (GSA) released a Notice of
Cancellation for FED-STD-209E, Airborne Particulate Cleanliness Classes
in Cleanrooms and Clean Zones, on November 29, 2001.
The Federal Standard 209E cancellation is based on the recommendation
by IEST Working Group CC100 that FED-STD-209E no longer be
maintained. The IEST, assigned by the GSA as the Preparing Activity
organization for FED-STD-209E, has recommended that International
Standard ISO 14644, Cleanrooms and controlled environments-Part 1:
Classification of air cleanliness, and Part 2: Specifications for testing and
monitoring to prove continued compliance with ISO 14644-1, supersede
FED-STD-209E. ISO 14644-1 and 14644-2 are two parts of a multi-part
group of ISO Standards developed by ISO Technical Committee 209
(ISO/TC 209)” (http://www.iest.org/Standards-RPs/ISO-Standards/FED-
STD-209E)
78

Manufacture….
FEDERAL STANDARD (FED-STD-209E)
 This standard discussed both conventional and unidirectional
cleanrooms.
 In the standard there is the first suggestion of measuring particles
of ≥ 0.5μm by means of optical particle counters; these
instruments had just become commercially available.
 It often asked why 0.5μm was adopted as the standard size on
which the Federal Standard was based. The answer is that it was
the ‘art of the achievable’, as this was the smallest size that was
easily measured by the particle counters available at that time.
80

Manufacture….
 It has been asked why 90 ft/min(0.45 m/s) was suggested in the
Federal Standard 209 as the velocity to be used in unidirectional
flow cleanrooms. It has been said that this was the velocity
theoretically calculated to remove a particle dropped in front of
the supply filter in the first laminar flow room at Sandia
Corporation.
81

Manufacture….
 In the older Federal Standards (A to D), the number of particles
equal to, and greater than 0.5 µm were measured in one cubic
foot(ft3) of air and this count used to classify a room.
 The most recent Federal Standard 209 (E version) accepted a
metric nomenclature, but in 1999, IS0 14644-1 was published.
This standard has been adopted by all countries in the European
Union and is now being adopted by other countries.
82

Manufacture….
The earlier Federal Standards 209 (A to D)
 The first Federal Standard 209 was publishedin 1963 in the USA
and titled “Cleanroom and Work Station Requirements, Controlled
Environments”.
 It was revised in 1966 (209A), 1973 (B), 1987 (C), 1988 (D) and
1992 (E).
 The cleanroom class limits, given in the earlier 209 A to D
versions, are shown in Table below.
 The actual class of a cleanroom is found by measuring the number
of particles ≥ 0.5 µm in one cubic foot of room air, and
determining which class limit is not exceeded; this is the
cleanroom classification.
83

Manufacture….
Federal Standard 209 D class limits
84

Manufacture….
Federal Standard 209 E
 In Federal Standard 209 E the airborne concentrations in the room
are given in metric units, i.e. per m3 and the classifications defined
as the logarithm of the airborne concentration of particles ≥ 0.5
µm per m3.
 For example, a Class M3.5 room has a class limit for particles ≥
0.5 µm of 3,530/m3.
 The logarithm of 3,530 is 3.5, which is the class. This is shown in
Table below.
85

Manufacture….
Federal Standard 209 E
86

Manufacture….
Federal Standard 209 E (summary)
class Particle size of
0.5µm–5.0µm per Ft3
Particle size of 0.5µm–
5.0µm per m3
United States (209E)
1 1 35.3 M1.5
10 10 353 M2.5
100 100 3530 M3.5
1000 1000 35,300 M4.5
10000 10000 353,000 M5.5
100,00
0
100,000 3,350,000 M6.5
Note that 1m3 is equal to 35.3 Ft3
87

IS0 Standard 14644
 The International Organization for Standards (ISO) is
developing a series of cleanrooms standards.
 These cover a wide variety of important cleanroom issues
such as design, testing, operation and bio-contamination.
 The first document, published in 1999, is IS0 14644-1 and
entitled “Part 1: Classification of air cleanliness”
 ISO 14644 is now composed of:
1. ISO 14644-1: Classification of air cleanliness
2. ISO/DIS 14644-1.2(2014): Classification of air cleanliness
by particle concentration
3. ISO 14644-2: Specifications for testing and monitoring to
prove continued compliance with ISO 14644-1
88

IS0 Standard 14644…
4. ISO/DIS 14644-2.2(2014): Monitoring to provide evidence of cleanroom
performance related to air cleanliness by particle concentration
5. ISO 14644-3: Test Methods
6. ISO 14644-4: Design, Construction, and Start-up
7. ISO 14644-5: Operations
8. ISO 14644-6: Vocabulary
9. ISO 14644-7: Separative devices (clean air hoods, gloveboxes,
isolators and minienvironments
10. ISO 14644-8: Classification of airborne molecular contamination
11. ISO 14644-9: Classification of surface particle cleanliness
12. ISO 14644-10: Classification of Surface Cleanliness by Chemical
Concentration
13. ISO 14644-12: Classification of Air Cleanliness by Nanoscale Particle
Concentration 89

IS0 14644-1 airborne particulate cleanliness classes for cleanrooms and clean
zones 90

Graphical representation of ISO-class concentration limits for selected IS0 classes
91

EU GMP classification
 The most recent pharmaceutical standard used in Europe came
into operation on January 1997.
 EU GMP guidelines are more stringent than others, requiring
cleanrooms to meet particle counts at operation (during
manufacturing process) and at rest (when manufacturing
process is not carried out, but AHU is on).
92

Airborne particulate classification of EU GMP for manufacture of sterile pharmaceutical
preparations
EU GMP classification…
93

 The particulate conditions given in Table above for the “at
rest” state should be achieved in the absence of the operating
personnel after a short “clean-up” period of about 15–20
minutes (guidance value), after completion of the operations.
The particulate conditions given in Table above for grade A “in
operation” should be maintained in the zone immediately
surrounding the product whenever the product or open
container is exposed to the environment.
EU GMP classification…
94

Comparison between selected equivalent classes of FS 209 and IS0 14644- 1
Comparison
NB: English in Ft3 metric ( in m3) is the International System of Units (SI)
95

Comparison of different airborne particulate classification systems for clean areas
Comparison…
96

WHO GMP classification
 For the manufacture of sterile pharmaceutical preparations, four
grades are distinguished by WHO, as follows:
Grade A:
 The local zone for high-risk operations, e.g. filling and making
aseptic connections. Normally such conditions are achieved by
using a unidirectional airflow workstation. Unidirectional airflow
systems should provide a homogeneous air speed of 0.36–0.54
m/s (guidance value) at a defined test position 15-30 cm below
the terminal filter or air distributor system.
 The velocity at working level should not be less than 0.36 m/s.
 The uniformity and effectiveness of the unidirectional airflow
should be demonstrated by undertaking airflow visualization
tests. 97

WHO GMP...
 Grade B: is the background environment for the grade A zone, in
aseptic preparation and filling.
 Grades C and D: Clean areas for carrying out less critical stages
in the manufacture of sterile products or carrying out activities
during which the product is not directly exposed (i.e. aseptic
connection with aseptic connectors and operations in a closed
system).
 In order to reach the B, C and D air grades the number of air
changes should be appropriate for the size of the room and the
equipment and personnel present in it.
98

Airflow Visualization Tests
 As part of the testing procedure to ensure that a cleanroom is
working correctly, the air movement within the room should be
checked. It is necessary to check that there is sufficient air
movement within the room to either dilute, or remove, airborne
contamination and hence prevent a build-up of contamination.
 There are a number of methods that can be used to visualize the
flow of the air in a cleanroom. These can be grouped as:
1. streamers
2. smoke or particle streams
99

Airflow…
1. Streamers
 The types of streamers that are used to visualize airflow are
threads or tapes. The best type are those which have a high
surface-area-to-weight ratio, and can be easily seen. Recording
tapes of the type used in music cassettes can be used, as can
loosely spun threads. A useful way of using streamers is to attach
it onto the end of an anemometer; it can then be used to ascertain
the direction of air when the velocity is being measured at a
particular spot.
100
Streamer

Airflow…
2. smoke or particle streams
 There are a number of methods suitable for generating smoke or
particle streams that are used to show the flow of air in
cleanrooms. For example, the apparatus used to produce smoke
for testing filter integrity can be used. However, the use of oil
smoke may not be acceptable in some cleanrooms, as oil left on
surfaces can be a contamination hazard. Water vapor is a
contamination-free alternative and can be produced by different
techniques such as from solid Carbon dioxide(dry ice) or by
nebulizing water.
101
Fogger producing water vapor
Smoke from a puffer

WHO GMP...
 The FDA defines two areas in aseptic processing that are of
particular importance to drug product quality. These are the
‘critical area’ and the ‘controlled area’.
 A ‘critical area’ is described in the FDA document as:
‘one in which the sterilized dosage form, containers, and
closures are exposed to the environment. Activities that are
conducted in this area include manipulations of these
sterilized materials/product prior to and during filling/closing
operations’.
102

WHO GMP...
 The ‘controlled area’ is described as:
‘an area in which it is important to control the environment, is
the area where unsterilized product, in-process materials, and
container/closures are prepared. This includes areas where
components are compounded, and where components, in-
process materials, drug products and drug product contact
surfaces of equipment, containers, and closures, after final
rinse of such surfaces, are exposed to the plant environment’.
103

WHO GMP...
Examples of cleanroom conditions required for different operations
104

 The area grades must be selected by the manufacturer on the
basis of the nature of the process operations being performed
and validation runs (e.g. sterile media fills).
 Appropriate alert and action limits should be set for the results
of particulate and microbiological monitoring. If these limits
are exceeded, the appropriate corrective actions should be
taken, as prescribed in the operating procedures.
WHO GMP...
105

 Components and most products should be prepared in at least a
grade D environment in order to give low microbial and
particulate counts, suitable for filtration and sterilization.
 Where the product is at unusual risk of microbial contamination
(e.g. because it actively supports microbial growth, must be held
for a long period before sterilization, or is necessarily not
processed mainly in closed vessels), the preparation should
generally be done in a grade C environment.
 The filling of products for terminal sterilization should generally
be done in at least a grade C environment.
Terminally sterilized products
106

 Where the product is at unusual risk of contamination from the
environment (e.g. 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.
Calculation of the likely airborne contamination
 If the exposed area of a product and the time that it is exposed to
airborne microbial contamination during manufacture is known,
then it is possible to calculate the product's contamination rate.
 Using the number of microbe carrying particles deposited on a
Petri dish in a given time and proportioning the areas and times
of exposure, the contamination rate can be calculated from the
following equation:
Terminally…
107

Example: 1
 A 14 cm Petri dish (154 cm2 area) is laid close to where
containers are filled and the microbial count on the settle plate
after four hours of exposure was 3 CFU. Calculate the number of
micro-organisms likely to deposit into the container of a neck
area of l cm2, when they are open during filling, for an average of
10 minutes.
Terminally…
108

Example: 2
 A Petri dish of 141 cm2 area is placed inside filling machine in
which PVC containers are filled and the microbial count on the
settle plate after 4 hours of exposure was 300 CFU. Calculate the
number of micro-organisms likely to deposit into the PVC bag of
a neck area of 0.283 cm2 when they are open during filling, for an
average of 7 seconds. (ans, 0.4 bags per batch/1,344 bags)
Terminally…
109

 The preparation and filling of ointments, creams, suspensions and
emulsions should generally be done in a grade C environment
before terminal sterilization.
 Components after washing should be handled in at least a grade
D environment. The handling of sterile starting materials and
components, unless subjected to sterilization or filtration through
a microorganism-retaining filter later in the process, should be
done in a grade A environment with a grade B background.
 The preparation of solutions which are to be sterile filtered during
the process should be done in a grade C environment; if not
sterile filtered, the preparation of materials and products should
be done in a grade A environment with a grade B background.
Aseptic preparation
110

 The handling and filling of aseptically prepared products, as well
as the handling of exposed sterile equipment, should be done in a
grade A environment with a grade B background.
 The transfer of partially closed containers, as used in freeze–
drying, should, before stoppering is completed, be done either in
a grade A environment with a grade B background or in sealed
transfer trays in a grade B environment.
 The 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 subsequently filtered.
Aseptic…
111

 Precautions to minimize contamination should be taken during
all processing stages, including the stages before sterilization.
 Preparations containing live microorganisms should not be made
or containers filled in areas used for the processing of other
pharmaceutical products; however, vaccines consisting of dead
organisms or of bacterial extracts may be dispensed into
containers, after validated inactivation and validated cleaning
procedures, in the same premises as other sterile pharmaceutical
products.
Processing…
112

Cleanroom classification vs air change rate
 Air changes per hour: are the number of total replacement of
any room’s air in one hour.
 Air changes per hour are a common way of expressing air
dilution in the room and give an indication of the cleanliness of a
room. However, the airborne cleanliness of a turbulently
ventilated cleanroom is really dependent on the volume of air
supplied to the room in a given time.
 If the supply of the air by HVAC system in one hour is equal to
the volume of the room then it will be one air change per hour.
 Rooms having 60 air changes per hour will have 60 times air
supply than the volume of the room.
Processing…
113

How to calculate air change per hour?
Air change/ hr=(average air flow velocity x area) of HEPA filter(s) x 60mi/hr
Room Volume(s)
Example
Assume that a certain cleanroom having a volume of 12m3, has one
HEPA filter of an area 0.36m2. The average air velocity of the HEPA
filter is 30m/min (average values taken from four corners and from
the center). Calculate the air change/hr.
Given
Average air flow velocity=30m/min
 Area of HEPA filter= 0.36m2
Room volume = 12m3
Processing…
114

Required
Air change/hour
Calculation
Air change/ hr=(average air flow velocity x area) of HEPA filter(s) x 60mi/hr
Room Volume(s)
Air change/ hr=(30m/min)(0.36m2)(60mi/hr)
12m3
Air change/hr= 54/hr
Processing…
115

Processing
Air velocities & change rate in cleanrooms
116

 validation of aseptic processing is Reading assignment
Processing…
117

 Water sources, water-treatment equipment and treated water
should be monitored regularly for chemicals, biological
contamination and contamination with endotoxins to ensure that
the water complies with the specifications appropriate to its use.
Records should be maintained of the results of the monitoring
and of any action taken.
Water for pharmaceuticals (USP36-NF31)
1.Drinking water 6. sterile water for injection
2. Purified water 7. sterile water for irrigation
3. Sterile Purified water 8. sterile water for inhalation
4. Water for injection 9. bacteriostatic water for injection
5.Water for hemodialysis
Processing…
118

 Activities in clean areas, especially when aseptic operations are
in progress, should be kept to a minimum, and the movement of
personnel should be controlled and methodical, so as to avoid
excessive shedding of particles and organisms due to over-
vigorous activity. The ambient temperature and humidity should
not be uncomfortably high because of the nature of the garments
worn.
Processing…
119

 The presence of containers and materials liable to generate fibres
should be minimized in clean areas and avoided completely when
aseptic work is in progress.
 i.e. Materials used in cleanrooms must be selected with care to
ensure that they do not contribute to the contamination in the
room. Typical of materials used in a cleanroom are:
 Materials used in the manufacturing such as containers and ingredients;
 Packaging for the product. For example, glass or plastic bottles, plastic bags and
pre-
formed boxes;
 Process machinery and equipment;
 Tools used for the maintenance, calibration or repair of equipment and machinery;
Clothing for personnel, such as suits, gloves and masks;
Materials for cleaning, such as wipers and mops;
Disposable items such as writing materials, labels and swabs.
Processing…
120

 Depending on the susceptibility of the product to Contamination,
some, or all, of the following list will be prohibited because of
contamination problems:
abrasives or powders;
aerosol-producing cans or bottles;
items made from wood, rubber, paper, leather, wool, cotton and other
naturally occurring materials that break up easily;
items made from mild steel, or other materials that rust, corrode or
oxidize;
items that cause problems when machined or processed, e.g. they
may smoke or break up;
paper not manufactured for use in cleanrooms. If ordinary paper must be
used then it should be sealed in plastic envelopes or laminated between
plastic films;
Processing…
121

 Components, bulk-product containers and equipment should be
handled after the final cleaning process in such a way that they
are not re-contaminated (The issue here is that the cleaned
process equipment does not stay clean indefinitely.).
 A drying step after cleaning can also help. Drying steps may
include flushing with an alcohol solution and allowing the alcohol
to evaporate or drying with a heated air or nitrogen purge.
Processing…
122

 The interval between the washing and drying and the sterilization
of components, bulk-product containers and equipment, as well
as between sterilization and use, should be as short as possible
and subject to a time-limit appropriate to the validated storage
conditions.
 It may involve qualification protocols in which equipment is
stored under worst case conditions for a defined time period after
cleaning. At the end of that time period, the equipment is tested
for re-contamination by suitable means. These suitable means
may include visual examination, microbial sampling, or testing
for chemical or particulate residues
Processing…
123

 The stage of processing of components, bulk product containers
and equipment should be properly identified.
 Use of operational status labels such as:
cleaned ,Under cleaning ,To be Ready for use, Under
maintenance, Off work for this time
Processing…
124

 The time between the start of the preparation of a solution and its
sterilization or filtration through a bacteria-retaining filter
should be as short as possible. A maximum permissible time
should be set for each product that takes into account its
composition and the prescribed method of storage.
Hold time (large volume parentrals)
1. Bulk solution before filtration and filling.
2. Filled solution in final containers before sterilization.
Processing…
125

 Time limits should be established for each phase of aseptic
processing.
 Time limits should include for example the period between the
start of bulk product, compounding and its filtration, filtration
processes, product exposure while on the processing line, and
storage of sterilized equipment, containers and closures.
 Bioburden and endotoxin load should be assessed when
establishing time limits for stages such as formulation processing
stage.
Processing…
126

 Any gas that is used to purge a solution or blanket a product
should be passed through a sterilizing filter.
 Sterilizing Grade Filter: is one that will produce a sterile effluent
after being challenged by microorganisms at a challenge level of
greater than or equal to 107/cm2 of effective filtration area.
Blanketing, Purging and Sparging
Blanketing: is the process of applying a gas to the empty space
in a storage container. The most common gas used in blanketing
is nitrogen.
Blanketing helps to protect products inside the storage container
and blanketing is used for a variety of products including cooking
oil, volatile combustible products(fuels), and purified water.
Processing…
127

Purging: A vessel’s headspace can be pressurized with nitrogen
to transfer liquids without the use of a pump. This can be
advantageous when pumping the material is difficult due to space
constraints or when the material, such as corrosives, can cause
pump issues.
Sparging: involves nitrogen being bubbled through or dissolved
into an oil, to force out oxygen or any other gases dissolved in it.
These are gases that can eventually replace the atmosphere above
it and lead to product degradation or unsafe condition
 sparging involves bubbling the formulation in the bulk
preparation vessels with gas (nitrogen).
Processing…
128

 The bioburden of products should be monitored before
sterilization. There should be a working limit on the
contamination of products immediately before sterilization that is
related to the efficiency of the method to be used and the risk of
pyrogens.
 Guidance limit for bioburden=NMT 10 CFU/100ml
 Guidance limit for Endotoxin=NMT 0.25IU/ml
Processing…
129

 All solutions, in particular large-volume parenterals, should be
passed through a microorganism-retaining filter, if possible
immediately before the filling process (pore size of 0.22µm).
Processing…
Cartridge filter
130

 Where aqueous solutions (in tankers) are held in sealed vessels,
any pressure-release outlets should be protected, e.g. by
hydrophobic microbiological air vent filter (also for WFI tank).
 Vent filters: are hydrophobic sterilizing-grade filters used as air
vents on processing tanks.
Processing…
Vent filter assembly
131

 Components, bulk-product containers, equipment and any other
articles required in a clean area where aseptic work is in
progress should be sterilized and, wherever possible, passed into
the area through double-ended sterilizers sealed into the wall.
Other procedures that prevent the introduction of contamination
(e.g. triple wrapping) may be acceptable in some circumstances.
 A double-ended sterilizer with interlocking doors with entry in
the laboratory and an exit in a clean area must be provided.
Processing…
132
Double-ended Sterilizers

 Extreme care must be taken with materials that have been
sterilized in the area for use in aseptic production, such as
primary containers and filling machine parts. After removal from
the sterilizer, they should be stored in a way which maintains
their sterility (examples: in laminair airflow, triple wrapping
etc.).
 All packs should be marked with the date of sterilization and
there should be a procedure setting out how long an item can
remain in the area before it needs to be re-sterilized.
 Certain components such as plastic materials that will not
withstand heat sterilization, in these case it is acceptable to
transfer pre-sterilized components into the aseptic areas and they
should be triple wrapped.
Processing…
133

 The efficacy of any new processing procedure should be
validated, and the validation should be repeated at regular
intervals thereafter or when any significant change is made in the
process or equipment.
Validation
 Validation: Documented evidence which provides a high degree
of assurance that a specific process will consistently produce a
product meeting its predetermined specifications and quality
attributes.
Processing…
134

 Will be discussed in module 2 (theory and practice of
pharmaceutical sterilization)
6. Sterilization
135

 The use of isolator technology to minimize human interventions
in processing areas may result in a significant decrease in the risk
of microbial 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 each zone can be realized.
 Isolators are constructed of various materials more or less prone
to puncture and leakage. Transfer devices may vary from single-
door to double-door designs to fully-sealed systems incorporating
sterilization mechanisms.
7. Isolator technology
136

 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 recognized that unidirectional airflow may not exist
in the working zone of all isolators and transfer devices.
 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.
Isolator…
137

 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, sanitization of the isolator, the transfer
process and isolator integrity.
 Monitoring should be done routinely and should include frequent
leak testing of the isolator and the glove/sleeve system.
Isolator…
138

 Isolators have been around the Pharmaceutical Industry since the
early 1980s and in the Nuclear Industry (glovebox technology)
since the 1950s.
 The intent of isolators is to create an airtight barrier or enclosure
around a piece of equipment or process which provides absolute
separation between the operator and product.
Isolator…
Isolator vs glove box
139

 The operator can perform tasks through half-suits or glove ports.
Isolators provide a specific environment inside the isolator using
HEPA filters.
 Reach inside the isolator is limited due to the length of glove
sleeves. When more reach or range of motion is required, a half-
suit is employed.
Isolator…
Half-suits Glove Ports 140

 The environment can be positive pressure or negative, can have
humidity control, use unidirectional airflow, and can either
protect the product from the operator as with aseptic processes, or
protect the operator from the product as with potent product
handling.
 The earliest uses of aseptic isolators were for sterility testing.
Isolator…
141

Isolators are constructed of various materials more or less prone
to puncture and leakage. Transfer devices may vary from single-
door to double-door designs to fully-sealed systems incorporating
sterilization mechanisms.
Isolator…
142

 Aseptic isolators can be constructed using both flexible materials
as well as rigid materials. Flexible wall isolators use clear plastic
film (usually PVC) at a variety of thicknesses. These isolators are
lighter weight, offer good visibility, and are easy to set up.
 Rigid wall isolators are generally made from 316L stainless steel
for the enclosure and laminated safety glass for viewing windows.
While these isolators are heavier and take more time to install,
they are more durable, do not absorb decontamination agents,
which result in fast decontamination cycles, and resist chemical
agents.
Isolator…
143

Isolator…
Plastic film isolator stainless steel isolator
144

APPLICATIONS
 Aseptic isolator uses include sterility testing, interface isolators,
transfer isolators, filling machine isolators, and powder
charging/handling isolators(handling of potent and hazardous
chemicals).
 Sterility test isolators are used to eliminate false positives during
the testing process.
 Interface isolators attach directly to a flange on the door of the
equipment being interfaced to, such as an autoclave,
depyrogenation oven, or lyophilizer.
 Due to the volume of the equipment chamber and the load within,
the isolator often utilizes a half-suit so the operator can reach into
the equipment and into any loading cart.
Isolator…
145

Isolator…
Interface isolators Sterility test isolators
handling isolator 146

Isolator validation and testing (reading assignment)
Reference
 <1208>Sterility testing-validation of isolator systems-USP36-
NF31
Isolator…
147

 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 or 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 sterilized should be
installed in at least a Grade D environment.
8. Blow/Fill/Seal Technology
148

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
sterilization-in-place;
 Background clean room environment in which the equipment is
located;
 Operator training and clothing; and
 Interventions in the critical zone of the equipment including any
aseptic assembly prior to the commencement of filling.
Blow/Fill/Seal…
149

 The B/F/S process enables a container to be formed, filled, and
sealed in one continuous, integrated operation using a single
automated machine.
 Blow/Fill/Seal (B/F/S) aseptic processing in parenteral
manufacturing enables the automated formation of a plastic
container, aseptic filling of the container with a liquid, and the
hermetic sealing of the container, all in a few seconds using one
machine.
Blow/Fill/Seal ..
150

 Because packaging of the formulated drug takes place under
aseptic conditions without any human intervention, it provides
increased product safety.
Blow/Fill/Seal…
Blow/Fill/Seal machine
151

Blow/Fill/Seal…
BFS Process
Thermoplastic resin is extruded into a
tubular shape called a parison
parison reaches the proper
length, the mold indexes,
pinching the bottom of the
parison closed. The top of the
parison is held open while the
parison is cut.
The mold is conveyed into
position under the
blowing/filling nozzle
assembly. The nozzle is
lowered into the parison,
forming a seal with the
neck of the mold. The
container is formed by
vacuum or assisted by
blowing with sterile
filtered air, expanding the
parison against walls of
the integrally cooled mold
cavity. While in position,
the sterile air is vented
from and sterile liquid
product is metered into
the container through the
fill nozzle.
The fill assembly retracts and
separate sealing molds close to
form the top, hermetically
sealing the container.
The mold opens and formed,
filled and sealed container is
conveyed out of the machine.
152

Resin Choices for B/F/S Processing
 LDPE, HDPE, P.P (Low, Medium and High Density)
 Polyethylene: Barrier properties improve as density increases,
clarity of container improves as density decreases.
 Polypropylenes: Excellent barrier properties and good clarity and
high temperature terminal sterilization.
Blow/Fill/Seal…
Products of BFS
153

 Only the minimum number of personnel required should be
present in clean areas; this is particularly important during
aseptic processes. Inspections and controls should be conducted
from outside such areas as far as possible.
 People can, when walking, produce about 1,000,000 particles ≥
0.5 µm and several thousand microbe-carrying particles per
minute. The more people, the higher the dispersion within the
cleanroom. It is therefore important that the minimum of people,
i.e. only the essential personnel are allowed into cleanrooms.
9. Personnel
154

 All personnel (including those concerned with cleaning and
maintenance) employed in such areas should receive initial and
regular training in disciplines relevant to the correct manufacture
of sterile products, including hygiene and 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.
 Visitors should be discouraged and only allowed in under the
control of a supervisor; if a cleanroom is designed with windows
for visitors to look into the cleanroom, this will usually suffice.
Personnel…
155

Personnel…
cleanrooms designed with windows for supervision
156

 Staff who have been engaged in the processing of animal-tissue
materials or of cultures of microorganisms other than those used
in the current manufacturing process should not enter sterile-
product areas unless rigorous and clearly defined
decontamination procedures have been followed.
Personnel…
157

 High standards of personal hygiene and cleanliness are essential,
and personnel involved in the manufacture of sterile preparations
should be instructed to report any conditions(e.g. coughs, colds
or any other type of infection) that may cause the shedding of
abnormal numbers or types of contaminants; periodic health
checks for such conditions are desirable. The action to be taken
in respect of personnel who might be introducing undue
microbiological hazards should be decided by a designated
competent person.
Personnel…
158

 Outdoor clothing should not be brought into changing rooms
leading to Grade B and C rooms. For every worker in a Grade
A/B area, clean sterile (sterilized or adequately sanitized)
protective garments should be provided at each work session.
Gloves should be regularly disinfected during operations. Masks
and gloves should be changed at least every working session.
Operators working in Grade A and B areas should wear sanitized
goggles.
 Wrist-watches and jewellery, should not be worn in clean areas,
and cosmetics, hair spray, nail varnish, etc. that can shed
particles should not be used.
Personnel…
159

Personal Items Not Allowed into the Cleanroom
 Food, drink, sweets and chewing gum
 Cans or bottles
 Smoking materials
 Radios, CD players, Walkmans, cell phones, pagers, etc.
 Newspapers, magazines, books and paper handkerchiefs
 Pencils and erasers
 Wallets, purses and other similar items.
Personnel…
160

 The clothing worn and its quality should be appropriate for the
process and the grade of the working area (workplace). It should
be worn in such a way as to protect the product from
contamination. The clothing required for each grade is as
follows:
 Grade D. The hair and, where relevant, beard and moustache
should be covered. Protective clothing and appropriate shoes or
overshoes should be worn. Appropriate measures should be taken
to avoid any contamination from outside the clean area.
Personnel…
161

 Grade C. The hair and, where relevant, beard and moustache
should be covered. A one-piece jumpsuit, gathered at the wrists
and with a high neck, and appropriate shoes or overshoes should
be worn. The clothing should shed virtually no fibres or
particulate matter.
Personnel…
162

 Grades A and B. Entry of personnel into Grade A areas should be
minimized. Headgear should totally enclose the hair and, where
relevant, beard and moustache. A one-piece jumpsuit, gathered at
the wrists and with a high neck, should be worn. The headgear
should be tucked into the neck of the suit. A facemask should be
worn to prevent the shedding of droplets. Sterilized, non-
powdered gloves of appropriate material and sterilized or
disinfected footwear should be worn. Trouser bottoms should be
tucked inside the footwear and garment sleeves into the gloves.
The protective clothing should shed virtually no fibres or
particulate matter and should retain particles shed by the body.
Personnel…
163

 Clothing used in clean areas should be laundered or cleaned in
such a way that it does not gather additional particulate
contaminants that can later be shed. Separate laundry facilities
for such clothing are desirable. If fibres are damaged by
inappropriate cleaning or sterilization, there may be an increased
risk of shedding particles. Washing and sterilization operations
should follow standard operating procedures.
Personnel…
164
Clothing for Grades A and B Clothing for Grades C and D

Personnel…
Garment Recommendations 165

Personnel…
Cleanroom garments
166

 All premises should, as far as possible, be designed to avoid the
unnecessary entry of supervisory or control personnel. Grade B
areas should be designed so that all operations can be observed
from outside.
 In clean areas, all exposed surfaces should be smooth,
impervious (not allowing liquid to go through) and unbroken in
order to minimize the shedding or accumulation of particles or
microorganisms and to permit the repeated application of
cleaning agents and disinfectants, where used.
10. Premises
167

 To reduce the accumulation of dust and to facilitate cleaning, there
should be no uncleanable recesses and a minimum of projecting ledges
(A horizontal projection forming a narrow shelf on a wall), shelves,
cupboards and equipment.
Premises…
Cleanroom
shelf
Cleanroom
cupboards
Cleanroom
cabinet
168
storage cabinet: wall
recessed

 Doors should be carefully designed to avoid uncleanable recesses;
sliding doors are undesirable for this reason. Swing doors should
open to the high-pressure side and be provided with self-closers.
Exceptions are permitted based on egress and site environmental,
health and safety containment requirements.
Premises…
Cleanroom Sliding Door Hinged Cleanroom Door
169
self-closers

 False ceilings should be sealed to prevent contamination from
void space above.
 Pipes and ducts and other utilities should be installed so that they
do not create recesses, unsealed openings and surfaces that are
difficult to clean.
 Sanitary pipes and fittings should be used and threaded pipe
connections should be avoided.
Premises…
False
ceilings
Pipes and
170

 Sanitary fittings and tubing (categorized also as hygienic
fittings and tubing) were developed and are used because they are
cleanable; either by dismantling a system and manually cleaning
it or using a CIP (clean in place) process.
Premises…
171

 Sinks and drains should be avoided wherever possible and should
be excluded from grade A/B areas where aseptic operations are
carried out.
 Where installed, they should be designed, located and maintained
so as to minimize the risks of microbiological contamination;
they should be fitted with effective, easily cleanable traps and
with air breaks to prevent back-flow. Any floor channels should
be open and easily cleanable and be connected to drains outside
the area in a manner that prevents the ingress of microbiological
contaminants.
 Drains should also be disinfected.
Premises…
172

 Drain trap: is designed to retain a small amount of water each
time the sink or drainage drains, and this water standing in the
bottom of the curved portion of the trap seals the drain and keeps
sewer gasses from escaping the drain and entering back to clean
areas.
 Air Break: is an indirect connection of a drain line to the drainage
system where the drain line terminates below flood level.
 Floor drain Channel: A trench drain (also channel drain, line
drain, slot drain, linear drain or strip drain) is a specific type
of floor drain containing a dominant trough- or channel-shaped
body. It is used for the rapid evacuation of surface water or for the
containment of utility lines or chemical spills.
Premises…
173

Premises…
Sinks Drains
174
drain trap
Air Break Floor drain Channel

 Changing rooms should be designed as airlocks and used to
provide physical separation of the different stages of changing
and so minimize microbial and particulate contamination of
protective clothing. They should be flushed effectively with
filtered air. The final stage of the changing room 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 sometimes desirable. In general hand-washing
facilities should be provided only in the first stage of the
changing rooms.
Premises…
175

 There should not be a change of more than one grade between
airlocks or passages and changing rooms, i.e. a Grade D passage
can lead to a Grade C airlock, which leads to a Grade B
changing room, which leads to a Grade B clean room.
 Changing rooms should be of a sufficient size to allow for ease of
changing. Changing rooms should be equipped with mirrors so
that personnel can confirm the correct fi t of garments before
leaving the changing room.
Premises…
176

Clothing change area
 Rooms used for changing into, and out of, cleanroom clothing
vary in design. The number of rooms in the change area, and
whether these rooms are divided into two or more zones by
crossover benches, will vary.
 The design of the change areas may also be complicated if
separate change rooms are provided for the two sexes.
Premises…
177
One-room change areas Two-room change areas with, or without, pass-over
benches

 Airlock doors should not be opened simultaneously. An
interlocking system and a visual and/or audible warning system
can be installed to prevent the opening of more than one door at
a time.
 A filtered air supply should be used to maintain a positive
pressure and an airflow relative to surrounding areas of a lower
grade under all operational conditions; it should flush the area
effectively.
Clean air supply to cleanroom
 There are two basic concepts of air supply/delivery to cleanroom
from air handling system (HVAC);
A recirculation system and
A full fresh air system (100% outside air supply).
Premises…
178

Premises…
Recirculation System
179

Premises…
Full fresh air system
180

 The type, number and placement of air supply diffusers, as well
as the extract/return grilles, is an important consideration in a
turbulently ventilated cleanroom. It is possible to supply the air to
a cleanroom with, or without, a diffuser.
 There may be alternative locations for return air, such as low-
level return air (preferable) and ceiling return air.
Premises…
181

Premises…
ceiling return
air
low-level return
182

 Adjacent rooms of different grades should have a pressure
differential of approximately 10–15 pascals (guidance value).
Particular attention should be paid to the protection of the zone
of greatest risk, i.e. the immediate environment to which the
product and the cleaned components in contact with it are
exposed.
 The recommendations regarding air supplies and pressure
differentials may need to be modified where it becomes necessary
to contain certain materials, e.g. pathogenic, highly toxic,
radioactive or live viral or bacterial materials or products. The
decontamination of the facilities and the treatment of air leaving
a clean area may be necessary for some operations.
Premises…
183

Premises…
A simple cleanroom suite showing pressures and aifflow between areas
184

 It should be demonstrated that airflow patterns do not present a
contamination risk; for example, care should be taken to ensure
that particles from a particle-generating person, operation or
machine are not conveyed to a zone of higher product risk.
 A warning system(usually audible) should be included to indicate
failure in the air supply. An indicator of pressure difference
should be fitted between areas where this difference is important,
and the pressure difference should be regularly recorded.
Premises…
185

 Consideration should be given to restricting unnecessary access
to critical filling areas, e.g. grade A filling zones, by means of a
physical barrier.
Premises…
186

11. Equipment
•Whenever possible, equipment used for processing sterile products
should be chosen so that it can be effectively sterilized by steam or
dry heat or other methods.
 A conveyor belt should not pass through a partition between a
grade A or B clean area and a processing area of lower air
cleanliness, unless the belt itself is continuously sterilized (e.g. in
a sterilizing tunnel).
A conveyor belt
187

 As far as possible, equipment fittings and services should be
designed and installed so that operations, maintenance and
repairs can be carried out outside the clean area. Equipment that
has to be taken apart for maintenance should be re-sterilized
after complete reassembly, wherever possible.
 When equipment maintenance is carried out within a clean area,
clean instruments and tools should be used, and the area should
be cleaned and disinfected again, where appropriate, before
processing recommences if the required standards of cleanliness
and/or asepsis have not been maintained during the maintenance
work.
Equipment…
188

 All equipment, including sterilizers, air-filtration systems, and
water-treatment systems, including stills, should be subject to
planned maintenance, validation and monitoring; its approved
use following maintenance work should be documented.
Types of equipment maintenances
1.Preventive or scheduled maintenance: where equipment or
facilities are inspected, maintained and protected before break
down or other problems occur.
2.Corrective maintenance: where equipment is repaired or
replaced after wear, malfunction or break down.
3.Predictive maintenance: which uses sensor data to monitor a
system, then continuously evaluates it against historical trends
to predict failure before it occurs
Equipment…
189

 Water-treatment plants and distribution systems should be
designed, constructed and maintained so as to ensure a reliable
source of water of an appropriate quality. They should not be
operated beyond their designed capacity. Consideration should
be given to including a testing programme in the maintenance of
a water system.
 Water for injection should be produced, stored and distributed in
a manner which prevents the growth of microorganisms, e.g. by
constant circulation at a temperature above 70°C or not more
than 4°C.
Equipment…
190

 Systems that operate and are maintained at elevated temperatures
(e.g. > 65ºC) are generally less susceptible to microbiological
contamination than systems that are maintained at lower
temperatures. When lower temperatures are required due to the
water treatment processes employed or the temperature
requirements for the water in use, special precautions should be
taken to prevent the ingress and proliferation of microbiological
contaminants.
Equipment…
191

 The headspace(non filled portion) in the storage vessel is an area
of risk where water droplets and air can come into contact at
temperatures that encourage the proliferation of microbiological
organisms. The use of spray-ball or distributor devices should be
considered in these systems to wet the surfaces during normal
operation, chemical and/or thermal sanitization.
Equipment…
headspacespray-ball
192

 Containers should be closed by appropriately validated methods.
Containers closed by fusion, e.g. glass or plastic ampoules,
should be subject to 100% integrity testing. Samples of other
containers should be checked for integrity according to
appropriate procedures.
12. Finishing of sterile products
193

 Containers sealed under vacuum should be sampled and the
samples tested, after an appropriate predetermined period, to
ensure that the vacuum has been maintained.
 Vacuum packing: is a method of packaging that removes air
from the package prior to sealing. This method involves
(manually or automatically) placing items in a plastic film
package, removing air from inside, and sealing the package.
 The intent of vacuum packing is usually to remove oxygen from
the container to extend the shelf life of product and, with flexible
package forms, to reduce the volume of the contents and package
(common in food industry).
 Vacuum packing reduces atmospheric oxygen, limiting the
growth of aerobic bacteria or fungi.
Finishing of…
194

Finishing of…
195
Vacuum packed food items

 Filled containers of parenteral products should be inspected
individually for extraneous contamination or other defects. When
inspection is carried out visually this should be done under
suitable and controlled conditions of illumination and
background.
 Operators doing the inspection should pass regular eyesight
checks, using personal corrective lenses (e.g. spectacles or
contact lenses) as required, and be allowed frequent breaks from
inspection. Where other methods of inspection are used, the
process should be validated and the performance of the
equipment checked at intervals. Results should be recorded.
Finishing of…
196

GMP Requirements for Sterile Products Manufacturing

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