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Prof.: Jacqueline Hernández, M.S.

Module Outline
 Scope
 Regulatory Requirements
 Aseptic Processing
 Microcontamination
 Types of Contamination
 Source of Contamination
 Personnel
Module Outline
 Equipment and Facilities
 Production Material and Processes
 Improper Work Practices
 Controlled Environmental Concepts

◦ Contamination Pathway
◦ Cleanroom Parameters
 ModuleOutline
 Personnel and Contamination Work

 Cleaning the Cleanroom

◦ Housekeeping
 Microbiology Laboratory
- Aseptic Techniques
◦ General Principles that should be
 CGMP’S Part 211
 Subpart C – Building and

 Subpart D – Equipment
 Subpart F - Production Controls

and Manufacturing
 Subpart I – Laboratory Controls
 Regulatory Requirements:
◦ <1211> Sterilization and
Sterility Assurance of
Compendial Articles
 Provides a review of the principles
involved in producing aseptically
processed products with a minimal
risk of microbial contamination in
the finished lot of final dosage
 Regulatory Requirements
◦ Chapter USP <1211>
◦ The areas of critical concern are the
immediate microbial environment
where these pre sterilized
components are exposed during
assembly to produce the finished
dosage form and the aseptic filling
 Regulatory Requirements
 Chapter USP <1211>

◦ The requirements for a properly

designed, validated and
maintenance filling or other
aseptic processing facility are
mainly directed to:
 an air environmental free from
viable microorganisms
 Regulatory Requirements
 Chapter USP<1211>

◦ Proper design to permit

effective maintenance of air
supply units
◦ Provision of trained operating
personnel who are adequately
equipped and gowned.
 Properlydesigned should have the
following considerations:
◦ Nonporous and smooth surface
 Including walls and ceilings
◦ Gowning rooms with adequate space for
personnel and storage of sterile
◦ Adequate separation of preparatory
rooms for personnel from final aseptic
processing rooms
 Regulatory Requirements <1211>
◦ Device as airlocks
◦ Air showers
◦ Proper pressure differentials between
◦ Employment of laminar (unidirectional)
airflow in the immediate vicinity of
exposed product or components.
 Regulatory Requirements
◦ Filtered air exposure with
adequate air change frequency.
◦ Appropriate humidity and
temperature environmental
◦ Documented sanitization program
 Regulatory Requirements <1211>
◦ Proper training of personnel in:
 Hygienic
Gowning techniques
 Gowns
Other body coverings substantially cover
exposed skin surfaces.
 Regulatory Requirements <1211>
◦ Certification and Validation of the
aseptic process and facility.
Efficiency of the filtration systems
Employing microbiological
environmental monitoring
Processing of sterile culture
medium as simulated product.
 RegulatoryRequirements <1211>
◦ Monitoring of the aseptic facility
Periodic environmental filter
Routine particulate examination
Microbiological environmental
Periodic sterile culture medium
 Regulatory Requirements <1211>
◦Sterility Testing
The facility should be such as to
offer no greater a microbial
challenge to the articles being
tested than that of an aseptic
processing production facility.
 Regulatory Requirements <1211>
◦ Sterility Testing
 The sterility testing procedure should be
performed by individuals having a high level
of aseptic techniques proficiency.
The test performance records of these
individuals should be documented.
 Cleanarea control
parameters should be
supported by microbiological
and particle data obtained
during qualification studies.
 It is important for area qualification and
classification to place most emphasis on
data generated under dynamic conditions
◦ Personnel present
◦ Equipment in place
◦ Operations ongoing
 US Federal Standard 209D
◦ Establishes standard classes of air
cleanliness for airborne particulate levels
in clean rooms and clean zones.
◦ Clean room class in the statistically
allowable number of particles, greater than
or equal to 0.5 micrometer in size, per
cubic feet of air.
 Definitions
 Isa room in which the
concentration of airborne
particles is controlled to
specific limits.
 They are classified by the
numbers of particles per
cubic foot per minutes.
 Sizes of particles:
◦ which are controlled usually range
from 0.1 micron to 10 microns.
 Typical room classes are Class 1,
10, 100, 1,000, 10,000 and
 Classes in ISO terms 1, 2, 3, 4, 5,

6, 7 and 8.
Acritical area is one in which
the sterilized drug product,
containers and closures are
exposed to environmental
conditions that must be
designed to maintain product
sterility (211.42(c)(10)
 This area is critical because an exposed
product is vulnerable to contamination and
will not be subsequently sterilized in its
immediate container.
 To maintain product sterility, it is essential

that the environment in which aseptic

operations area conducted be controlled
and maintained at an appropriate quality.
 One aspect of environmental quality is the
particle content of the air.
 Particles are significant because they can

enter a product as an extraneous

◦ and can also contaminate it biologically by acting
as a vehicle for microorganisms.
 Class Limits in Particles per Cubic Feet

Room 0.1 0.2 0.3 0.5 5.0

1 35 7.5 3 1 n/a
10 350 75 30 10 n/a
100 n/a 750 300 100 n/a
1000 n/a n/a n/a 1000 7
10,000 n/a n/a n/a 10,000 70
100,00 n/a n/a n/a 100,00 700
 The nature of the activities conducted in a
supporting clean area determines its
 FDA recommends that the area

immediately adjacent to the aseptic

processing line meet, at a minimum, Class
10,000 (ISO 7) standards under dynamic
 Manufactures can also classify
this area as Class 1,000 (ISO 6).
 An area classified at a Class
100,000 (ISO 8) air cleanliness
level is appropriate for less
critical activities
◦ Equipment cleaning
Material which we do not
There are a multitude of
material and processes both
biological and industrial, that
are sensitive to contamination.
 An essential part of contamination
prevention is the adequate
separation of areas of operation.
 To maintain air quality, it is

important to achieve a proper

airflow from areas of higher
cleanliness to adjacent less clean
 It is vital for rooms of higher air cleanliness
to have a substantial positive pressure
differential relative to adjacent rooms of
lower air cleanliness.
 For example, a positive pressure differential

of at least 10-15 Pascals (Pa6) should be

maintained between adjacent rooms
differing classification (with doors closed).
 When doors are open, outward
airflow should be sufficient to
minimize ingress of
contamination, and it is critical
that the time a door can remain
ajar be strictly controlled.
 The Agency recommends that pressure
differentials between cleanrooms be
monitored continouosly throughout each
shift and frequently recorded.
 All alarms should be documented and

deviations from established limits should

be investigated

It may be obvious to us when it
is in the form of dust and lint on
a work surface.
It may be in the less
distinguishable form of vapor in
the air.
Types of Contamination
 Organic
 Inorganic Substances
 Inorganic Materials
 Can be natural
 or Man made solids, liquids or gases
They can be elemental = consisting of one
elements of the periodic table.
Compounds = chemical combinations of two
or more elements (ex. Sodium chloride)
Mixtures = such as metal alloys (ex. Brass)
Organic Contaminants
 Complex chemical structures are
typical of organic contaminants if
they are of biological origin.
 Three Categories:
 Particulate Materials
 Bacteria, fungi or other materials
generated by biological processes
 Gases and vapors

Particulate Materials
Consist of metal fragments, lint
from clothing, skin flakes and
human hair.
Clearly every effort must be
made to reduce the number of
these particles to an absolute
 Bacteria, Fungi…
 Bacteria and fungi can grow on a
wide variety of material in light and
The residue of these growth
processes can be highly toxic or
sufficiently alkaline to each material.
 We can limit their rate of growth to
the point where they do not present
a severe problem.

Gases and Vapors
 Condensed organics can
serve as growth media for
bacteria or fungi.
 Variety of materials can
evaporate and then
condensed as films or

Airborne Particulates
Contamination is usually in small
quantities compared to whatever
is being contaminated.
Solid contamination typically will
take the form of small particles in
a range of sizes.
 Airborne Particulates
Visible particles are in the order of
50 microns.
Bacteria range from 0.3 microns.
Face powder is in the range 0.5
micron to 10 micron.
Smoke ranges from 0.01 micron to
1 micron.
 Particles
may be transported to our
sensitive site by:
Process chemicals
On the surfaces of process solids
 Airborne Particulates
Once particles are generated and
released from their source
materials, they may be carried by
air currents generated by
meteorological conditions:
Movement of people
Movement of equipment
Gravitationalforce = mass x
acceleration due to gravity
The drag forces are not so
easily represented.
Depend on a number of factors
such as the density and velocity of
the air and the nature of the
Airborne Particulates
 Near-spherical particles greater
than 1 micron in diameter will
settle quite quickly under the
force of gravity.
 This is important will result in the
removal of around 99.9% of these
larger particles from still air.

Airborne Particulates
 Particles smaller than 1
micron but larger than 0.1
micron in diameter will
settle very slowly.
 will be carried readily by
even slight air movements.

Airborne Particulates
Particles less than 0.1
micron in diameter still
totally in air.
These particles will tend to
remain suspended almost
 there are no moving air drag

Airborne Particulate
 The mechanisms here involves
the motion of the gas molecules.
In air at room temperature the
gas molecules are moving with
an average velocity in random
 Depend on temperature

Brownian Motion
The particles “dance
around” in the still air
as they are hit from all
sides by the molecules.

Gases and Vapors
Gaseous contamination
may also be transported in
moving air by:
 mixing
 in still air by diffusion

Gases and Vapors
Transport due to mixing will
depend on the velocity and
turbulence or the flow.
Transport due to diffusion
depends on the diffusion rate of
the gas in air.
Depends on the type of gas and
the concentration gradients.

Gases and Vapors
 Vapors may react from the
vapor phase, but they may also
condense as a thin layer of
liquid or semisolid on the
Example: oil vapors may
recondense on a cool surface to
create a thin oil film.
Contamination in Liquids
If particles are created of fall into
a liquid, they may be readily
transported by that liquid in the
same way as air transport them.
Liquids transport larger particles
than air at the same velocity.
Contamination in Liquids
A further problem with particles
in liquids is that the particle
material may be soluble to some
extent in the liquid.
Ex. Many minerals dissolve in water
and some organic substances in
Contamination in Liquids
This disperse the contamination
and makes its removal very
Gases and vapors may also
become dissolved in liquids.
Unwanted liquid materials may
also contaminate liquids
Ex. Alcohol in water
Contamination in Liquids
 Liquid contamination need
not mix with the water to be
 Ex. Most oils do not mix water
but will be carried with it.
 One highly significant
source of contamination
internal to the production
environment is the
personnel who work in
that environment.
 Personnel
 Personnel are critical for two very
good reasons:
Humans are animals and as such are
biological contamination “factories”
The operators are frequently the
closest thing to the actual elements we
wish to keep free from contamination.

“Dirty People”
Cleanest people produce huge
amounts of contamination as
part of the process of living.
 Personnel
 The most significant contamination

produced by humans is skin flakes.

 The skin we are covered with is there to

protect us from the environment we live in.

 We are regularly bombarded by harmful

ultraviolet rays which can actually destroy

cells and tissue.
 Personnel
The human body defends itself by sacrificing its
outermost layer of skin and allowing it to become
The dead cells flakes off when the
skin is gently abraded and will be
carried off by:
the convection currents surrounding
the body
or channeled through the clothing.

 The cells are organic
 They are complex in a chemical
 They carry other substances which
are naturally found on the skin.
Ex. Sodium chloride from perspiration
 skin oils

The number of skin flakes
particles shed depends:
 the state of the skin
 the activity of the subject
 If the skin is moistened by
perspiration or by the
application of a moisturizing
skin cream, the skin flakes
will tend to stick to the
surface better.

Body heat will result in the
drying out of the skin and
the subsequent release of
the cells.
Dry cells will also tend to
fracture into many small
 Move Slowly and Deliberately
◦ Rapid movements can create unacceptable
turbulence in a critical area.
◦ Such movements disrupt the unidirectional
airflow, presenting a challenge beyond intended
cleanroom design and control parameters.
 Move Slowly and Deliberately
◦ The principle of slow, careful
movement should be followed
throughout the cleanroom.
◦ Keep the entire body out the path
of unidirectional airflow.
 Personnel
 Move Slowly and Deliberately
◦ Unidirectional airflow design is used to
protect sterile equipment surfaces,
container closure and product.
◦ Disruption of the path of unidirectional
flow air in the critical area can pose a
risk to product sterility.
 Move Slowly and Deliberately
◦ Approach a necessary manipulation
in a manner that does not
compromise sterility of the product.

A good rule of thumb is that
a motionless person will
generate some 100,000
particles of approximately
0.3 to 0.5 microns diameter
per minute.
 Personnel
This figure changes dramatically
when the subject begins an activity.
As the increased abrasion of clothing
against skin and limbs against body,
As the constant expansion and
contraction of the flexible skin will
result in the expulsion of a greater
number of particles.

A person sitting at a
workbench moving just the
arms and body generate on
average 1,000,000 particles
per minutes.
If the whole body is in
motion, for example
during walking, this
number increases by at
least a factor of five.
 Personnel
Skin flakes are not the only
contamination that the human body
Small flakes of the protein keratin
are released from the hair.
Hair itself will also be released from all
over the body.
 Personnel
The act of breathing produces a
great many contaminants.
 In addition to large quantities of
carbon dioxide and water vapor.
 there will be droplets of water with
dissolved salts and suspended
biological material
 ex. Parts of cells from the inside of the

This respiration-related
contamination can be projected
large distances from the body
Carried by the exhaled air
A cough or sneeze can projected
the contaminants at high velocity
onto critical surface.
Eyes can cause problems.
The tear ducts regularly produce
a saline solution to wet the eyes.
When we blink, small droplets
are splashed out of the eyes.

 Unfortunately, we
occasionally make the human
contamination factor worse
by adding to the materials
which may be shed from the
surface of the skin.
 Example:
Most cosmetics have a powder base
which is mixed with an appropriate
liquid carrier for ease of application.
Once this carrier evaporates, we are
left with a semi-dry material which will
either flakes off by itself or be carries
on skin cell particles.

Mascara and other eye
cosmetics contain large
amounts of fine carbon particles
which can be shed with time.
The colorants in cosmetics are
a veritable periodic table with
such elements as zinc and iron
regularly appearing.
 Personnel
Men also wear powder-based
deodorants and antiperspirants, the
latter containing aluminum
The use of aftershave is also
problematic, as the alcohol content
will dissolve the natural skin oils
and thereby allow easier shedding.
 Personnel

◦ A well designed, maintained and

operated aseptic process minimizes
personnel intervention.
◦ As operator activities increase in an
aseptic processing operation, the
risk to finished product sterility also

◦To ensure maintenance of

product sterility, it is critical
for operators involved in
aseptic activities to use
aseptic techniques at all