GMP requirement to control

microbial contamination in non

sterile product

(Includes personnel, production, facility, utility,

equipment)

Glossary
At rest
A term for the static state with all services operating, but without personnel present.
Bioburden
The total microbial load with which an object or sample is contaminated.
Biofilm
An organized microbial system consisting of layers of microbial cells associated with surfaces,
often with complex structural and functional characteristics. Biofilms have physical/chemical
gradients that influence microbial metabolic processes. They can form on inanimate devices and
also cause fouling .
CFU (Colony Forming Unit)
Viable micro-organisms (bacteria, yeasts and mould) capable of growth under the prescribed
conditions (medium, atmosphere, time and temperature) develop into visible colonies (colony
forming units) which are counted. The term colony forming unit (CFU) is used because a colony
may result from a single micro-organism or from a clump/cluster of micro-organisms. It is
normally expressed as CFU per g or mL.
Cleaning
The removal of soil from a surface.
Clean room
A room in which the concentration of airborne particles is controlled to a defined standard. This
is achieved by controlling the introduction, formation and retention of particles.
Contact time
The total time an organism is exposed to the antimicrobial action of a disinfectant.
Contaminant
A foreign agent not introduced as part of processing, such as airborne particulates or
adventitious micro-organisms.
Dead leg
Any length of pipework that does not allow effective circulation of its contents, resulting in the
potential for contamination.
Disinfectant
An agent that reduces the level of micro-organisms to one that is safe for the relevant purpose.
Usually a chemical agent but sometimes may be a physical one such as X-rays or ultraviolet light.
Dynamic state
Refers to environmental or particle monitoring when a room is occupied by personnel.
FMEA (Failure Mode and Effects Analysis)
A risk assessment and risk mitigation tool.
HACCP (Hazard Analysis and Critical Control Points)
A formalized process by which a manufacturer ensures that all steps critical to product safety are
assessed and that adequate safety procedures are identified, implemented and reviewed.
HEPA (High Efficiency Particulate Air)

GMP requirement to control Microbial contamination in non sterile products

2014

Describes the system for filtering (diluting) air into clean-rooms. Standard HEPA filters remove
99.97% of 0.3m particles.
Medium (plural, media)
Any liquid or solid material prepared for the growth, maintenance, or storage of microorganisms.
Microbiological Control
The sum total of activities undertaken to manage and mitigate risks from microbiological sources.
Microbiological Monitoring
Sampling, testing, trending and reporting activities undertaken to assess and understand the
state of microbiological control.
Objectionable organism
An organism that is risk assessed to be objectionable with respect to its potential impact to
patients. Some objectionable organisms are specified in the pharmacopoeias but these are not
exclusive and other organisms may be objectionable depending on the nature of the product,
route of administration and intended patient population.
Out-of-Specification (OOS)
An OOS result is one which falls outside the specifications or acceptance criteria established in
new drug applications, official compendia, or by the manufacturer within their internal
procedures, e.g. Action levels for environmental monitoring data.
Out-of-Trend (OOT)
With respect to environmental monitoring data, the exceeding of an alert level or a defined
statistical signal.
RODAC (Replicate Organism Detection And Counting)
The international acronym for a contact plate (that is an agar plate, with a raised surface, applied to a
surface for the enumeration of micro-organsims).
Risk assessment
A systematic process of organizing information to support a risk decision to be made within a risk
management process. It consists of the identification of hazard and the analysis and evaluation
of risks associated with the exposure to those hazards (ICH Q9).
Settle plate
An agar plate used for passive air-sampling. The plate is exposed for a fixed duration, after which
it is incubated, and the number of micro-organisms which have settled on it and have produced
colonies are counted.
Sporicide
An agent that destroys microbial spores, especially a chemical substance that kills bacterial
spores.
Water activity (aw)
A measurement of the energy status of the water in a system. It is defined as the vapor pressure of
water above a sample divided by that of pure water at the same temperature; therefore, pure distilled
water has a water activity of exactly one.

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GMP requirement to control Microbial contamination in non sterile products

2014

There are a number of potential sources of

microbial contamination within a pharmaceutical
facility, including:
1. Personnel

2. Air/compressed
gases

5. Equipment

6. Water

3. Process
design

Facility
design and
layout
7. Product formulation
8. (active, starting
materials, packaging
components)
4.

There are a number of potential

sources of microbial contamination within a pharmaceutical facility,
including:
1-

Personnel

1.1.

All individuals who enter the production areas at any time.

Access to production areas must be controlled to ensure
entry is restricted to trained personnel and accompanied
visitors.

GMP Training:

Training of below mentioned topics

Introduction to micro-organisms and microbiological contamination control
Entry and exit of production facilities (including gowning)
Personal hygiene training
Good working practices

1.2.

Hygiene

For non-sterile manufacture the hygiene program will include:

Medical examination upon recruitment
A procedure for the notification of health conditions that may affect product
quality and subsequent actions to prevent contact with starting materials,
primary packaging materials and manufactured products by those personnel
with:
1- Infectious disease
2- Open lesions on any exposed part of body
3- Shedding skin conditions, such as eczema or psoriasis
4- Objectionable micro-organisms that may impact pharmaceutical
products and patients)
5- Gastric upsets
6- Steps to be taken to prevent direct contact between the operators
skin and any pharmaceutical product/product contact equipment.

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GMP requirement to control Microbial contamination in non sterile products

1.3.

The prohibition of eating, drinking, chewing or smoking, or the storage of food, drinks,
smoking materials or personal medication in the production or storage areas
A requirement for personnel to wash their hands before entering the manufacturing
facility

Gowning
In general, a non-sterile manufacturing environment will require
Wearing of suitable protective over-garments appropriate to the

2-

2014

operations to be carried out generally one piece over-garments are often

used as they provide a better level of protection to both the operation and
the product
Wearing a hair cover (e.g. mob cap), beard or moustache cover (where
applicable) and overshoes.
Dedicated shoes are recommended for personnel working in product ion
areas for long periods
Requiring the removal of jewelers, in particular rings with stones and

earrings, and make up

Gloves must be worn when performing production activities (including the
handling of components) to ensure there is no contact between operators
hands and exposed products or any part of equipment that comes
into contact with product.
Visitors not entering production rooms may only require a laboratory coat,
rather than a one piece over-garment.
Personnel and material flows should be considered when planning the
facility and processes. Additional care should be taken with staff movement
and production scheduling.

Facility and Utility

Facility and equipment design cannot eliminate
microbial contamination.
But good hygienic design can mitigate several of
these risks.
For example, well designed water and HVAC
systems minimize microbial ingress and aid
monitoring; equipment designs with smooth
surfaces of appropriate materials can facilitate
effective cleaning.

2.1. Material Flow

Personnel access must only be made via changing rooms

Access to the facility must be restricted to personnel that are fully trained and
assessed as competent to work in that area.
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GMP requirement to control Microbial contamination in non sterile products

2014

All visitors should be accompanied at all times by fully trained

personnel.
The changing room design contributes to the assurance of
appropriate personnel access and microbial contamination control.
The requirements for area entry must be written into procedures:
o It is usual to have pictures of correctly clothed personnel
o Mirrors to enable staff to check them before entry.
o The changing room should be provided with filtered air.
o It is useful to have a step over line or bench to define the
boundaries of clothing requirements.
o Hand washing facilities should be included in the design of
changing rooms and used prior to access to the production areas.
A copy of the gowning procedure should be present in the changing room.

2.2 Building requirement

2.2.1. Surface
Surfaces should be:
Smooth that will not harbor micro-organisms and can withstand the routine
cleaning regimes required.
Floors should be:
Constructed of durable cleanable materials with minimal joins and will typically be
epoxy resin or sheet vinyl, not vinyl tiles.
Lights should be
Encased, flush with the ceiling or wall panels and appropriately
sealed with silicone to prevent collection of dust and microbial
matter
Coving or moulding should be:
Used at the junctions between floor and wall and between ceiling
and wall to eliminate right-angle joints and aid cleaning. The joints should be
sealed with silicone and the coving maintained in a good condition to prevent
collection of dust and microbial matter
Sinks within the production areas should be:
Made of stainless steel and designed to be self-draining to minimize any
pooling or trapping of water and to maximize cleanability
Benches and trolleys should be:
Made of cleanable, impermeable materials and have smooth surfaces.
The use of mobile benches can facilitate cleaning. However, where mobile units
are used, consideration must be given to maintaining cleanliness of the wheels,
e.g., through the use of sticky mats between rooms.
At points of entry to the facility or to rooms, sticky mats or polymeric flooring
areas can be beneficial in the prevention of cross-contamination by removing
particulate material from the soles of shoes or wheels. Windows/viewing
panels should be non-opening, encased, and flush with the wall panels and
appropriately sealed to prevent collection of dust and microbial matter.
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GMP requirement to control Microbial contamination in non sterile products

2014

2.2.2. Drainage
As drains are a potential source of contamination, they should be minimized.
Where used, they should be
Hygienically designed to reduce the risk of aerosols, standing water and splash
back.
Drain covers, surfaces and joints should be:
Smooth and appropriate containment features should be incorporated within the
design.
Water from any process system must have:
An air break into a tundish before it is piped away conventionally to a floor
drain.
The floor drain will be trapped but there is still potential for the water in the drain to
be contaminated. The floor drain should be connected via a break tank (as minimum)
to the foul drain system. Although it does allow for the containment of contaminated
spillage (preventing it reaching the local foul system), the primary purpose of the
break tank is to prevent any backflow of contaminated waste from the foul drain into
the process drainage system.
Drains is required to be
Disinfected on a regular basis upon a known understanding of the microbial
contamination risk presented because drains are the primary link between the
room and the external drainage system
2.2.3. Heating, Ventilation and Air Conditioning (HVAC) System
Humidity control is key for some storage areas if materials are hygroscopic, increased
moisture levels may present a risk to microbial proliferation.
Air quality is improved by filtering the air through progressively more efficient filter
media.
1. Higher risk activities are recommended through the industry to use
Absolute filtration, using High Efficiency Particulate Air (HEPA) filters
removing 99.97% of particles sized greater than 0.3m
2. However, in lower risk activities e.g. tablet manufacture less efficient
filters, e.g. bag filters removing 90% of particles sized greater than
0.5m, with turbulent air flow are sufficient.

Current industry practice is to have:

1- Pressure differentials of 1020 Pa between areas of different standards.
2- Air changes for lower risk activities typically 1015 per hour.
Air changes rates for higher risk activities are higher, typically greater than 20 per
hour.
3- An appropriate Operational practices. For example, where a processing room is
at negative pressure to a corridor for the containment of active ingredients, there is an
increased risk of microbial ingress from the corridor. Consequently, an effective
cleaning regime for the corridor is required to ensure that microbial levels are kept
low.
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GMP requirement to control Microbial contamination in non sterile products

2014

Table of different standards for air classification

EU GMP
ISO 14644
US federal
Annex 1 2008 At rest
1999 pt1
Standard 209E Withdrawn
A-B
5
100

0.5m
Particles/m3
3520

5m
Particles/m3
Grade A=20
Grade B= 29

10000

352000

2900

100000

3520000

29000

2.3. Utilities
2.3.1. Compressed gases:
Filtered at end of use and measuring differential pressure before and after the filter.
2.3.2. Water
2.3.2.1 Potable Water
Potable water may be used for hand washing and initial cleaning or wash-inplace systems.
2.3.2.2 Purified water:
Purified water is generated from potable water, e.g. by
1. Reverse osmosis or
2. Distillation, though the latter is typically only used for the generation
of WFI
Where process water is treated by the manufacturer to create the
appropriate quality, the treatment process should be validated and
routinely monitored.
Incoming water should be sampled at point of entry on a routine basis.
Piped purified water systems are typically monitored
1. Continually on-line for conductivity and Total Organic Carbon (TOC)
2. Routinely for chemical and microbial quality.
Most microbial problems arise from the storage and distribution of the
water rather than its generation the development of biofilms can be extremely
difficult to resolve.
However, improperly maintained generation components, e.g. carbon beds,
softeners, reverse osmosis membranes, can also contribute to subsequent
contamination downstream within the distribution system.

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GMP requirement to control Microbial contamination in non sterile products

2014

Various design features may be utilized to prevent the development of

biofilms and control contamination:
1- Continual circulation with adequate flow rate (typically 13 m/s) to aid the prevention of
biofilm formation and minimize dead legs.
2- The capacity to heat the water to elevated temperatures (typically 6585C) for
sanitization purposes
3- Inclusion of high intensity UV lamps. The inclusion of UV lamps downstream of potential
microbial reservoirs, e.g. carbon beds, softeners, has the added advantage of enabling
ozone to be used for sanitization
Where UV lamps are used they should be regularly checked and maintained to ensure
they are clean and provide the correct wavelength and energy output
The inclusion of filters within the distribution loop is difficult to justify and is not advisable.

Older systems, without such design features may require regular disinfection using
an oxidizing agent to control biofilm e.g. ozone, hydrogen peroxide, hypochlorite

Each of these methods has disadvantages, e.g. additional design considerations (UV light
to destroy ozone) or Health and Safety risks, and none is fully effective. Extensive flushing
is required to remove chemical residues if hypochlorite is used, which is expensive and
disruptive.
Typically, stainless steel pipe work will be used, though systems with plastic piping have
also been used.
Sanitary design for valves is an expectation.
Operating procedures should require outlets to be flushed before usage To:
Ensure use of the circulating water
Remove possible stagnant water or contamination from the surface of the outlet.
The flushing of outlets prior to sampling for monitoring purposes should be
equivalent to that applied in operational use
The use of hoses and temporary piping is a major source of contamination
Where used they should be
to product in non-sterile facilities and therefore their use should be
subject to appropriate
controls to minimize the risk of
minimized.
contamination from this
2.3.3. Steam
source. E.g., they should
Clean steam is used for
1.
2.
3.

Cleaning and sanitization of production tools and equipment,

Supply for autoclaves and
Humidification of air where required.

The microbial risk from the steam itself is low due to the physical
characteristics of steam and its production from purified water using a heat
exchange.
Care should be taken with respect to condensation generated upon cooling
of the steam on surfaces, which may be sources of contamination that may
then be spread by the condensate.

1. Not be left on the outlets

2. Dried after use,
3. Hung vertically in appropriate
locations to ensure free
drainage,
4. Monitored and regularly
cleaned,
5. Sanitized and replaced.
6. Samples for microbial

monitoring should be done

including the used hoses.
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GMP requirement to control Microbial contamination in non sterile products

4-

Cleaning and
Disinfection:

2014

Sufficient time and resource should be allocated for cleaning activities.

4.1. Cleaning Documentation system

Areas must be regularly cleaned and, where

necessary, disinfected to a written procedure
and programmed.
Standard Operating Procedures (SOPs) are
required and need to be part of the document
control system.
SOPs need to state the
Areas to be cleaned and their
frequency,
Materials, equipment and methods.
A cleaning log is required which records the areas cleaned, agents used, type of
cleaning (as appropriate) and the identity initials of the operator.

4.2. Frequency

The frequency of different cleaning and disinfectant activities will vary depending on
the risk assessment (which takes account of room usage and formulation).
For example, floors, fittings and benches may have a routine daily clean, while higher
level walls and ceilings may be cleaned less frequently, e.g. weekly or monthly.

4.3. Sequence and Method

The method and sequences of cleaning is important; contact time, application

temperature, mechanical action and the chemistry of the cleaning agents should all be
considered during the design of the cleaning process.
Where used, all residual cleaning and sanitizing agents must be removed from
product contact surfaces to avoid product contamination
Fittings and fixtures including door handles, light switches and telephone receivers
should be included in the cleaning regime
Water should never be left to stand in the buckets but used and immediately
discarded to ensure that a potentially significant source of microbiological
contamination is removed as soon as possible.
All items, including machines used for mechanical cleaning, need to be stored clean
and dry.
There should be a defined drying method/area and storage area, room or cupboard
for dried cleaning utensils and materials because residues of liquid and moisture
encourage microbial growth and the development of resistant strains.
It is important that all cleaning regimes (automated and manual) are fully validated
or subject to verification to ensure that the equipment used for pharmaceutical
production is free from residues of product or cleaning material and microbial
contamination.

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o Hot water or hot water with

detergent followed by a hot
water rinse may be all that is
necessary in some areas.
o Whereas other facilities will
require detergent clean
followed by a disinfectant.
This should be determined
and documented by the risk
assessment.

GMP requirement to control Microbial contamination in non sterile products

2014

4.4. Cleaning agents

Steps for preparing the correct concentrations are particularly relevant

Disinfectant rotation is a contentious issue attracting considerable debate
within industry and regulatory agencies. However, this is specific to sterile
manufacture.
The principle scientific argument for rotation is to ensure activity against the full
spectrum of flora within the facility; one category of agent may not work against all
types of organisms (e.g. sporicides for use against spore forming organisms).
Disinfectants should only be applied to relatively clean surfaces since they can be
inactivated by residues of dirt and other materials.
Detergents are primarily used for the removal of soil/dirt residues whilst
disinfectants are primarily targeted at microbiological control.
All cleaning agents should be freshly prepared with water of a suitable quality
(typically purified water, although potable water may be acceptable).
Cleaning agents and prepared solutions should state the dates of preparation and
expiry clearly on the container.

Dirty Equipment Hold Time (DEHT) is the time between the end of manufacture and
cleaning.
The DEHT should be kept to a minimum because over time product residues may
become harder to remove and the risk of microbial cross-contamination increases.
Maximum times should be defined and supported by validation.
Clean Equipment Hold Time (CEHT) is the time from cleaning to next use.
CEHT is important to assure equipment does not become re-contaminated during
storage either by microbial contamination or dust.
The CEHT should be a defined and documented period up to which the cleanliness of
the equipment has been demonstrated. This may be achieved through a hold time
study, i.e. validated

Biofilm development is a risk with some items of equipment and must be considered
as part of risk assessment both in relation to design and cleaning processes.
Where biofilms do occur, remediation can be difficult and contingency plans for
dealing with them should be considered.

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GMP requirement to control Microbial contamination in non sterile products

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Formulation
Risk management during manufacturing
Important factors to consider in the
manufacturing process include:

5.1. Processing steps

5.1.1 Solvents used:
Water-based processes provide a more
favorable environment for
microorganisms
The use of other solvents might decrease the risk of microbial growth.
5.1.2 Temperatures used:
2535C promoting microbial growth.
Significantly higher or lower temperatures being detrimental to micro-organisms
5.1.3 Hold times and overall campaign length:
Longer processing times may increase the opportunity for microbial proliferation
unless the conditions are detrimental to microbial growth
Processes requiring heavy physical labor or increased numbers of personnel might
increase the risk of contamination from personnel through enhanced sweating or
shedding of skin particles.
Water is a frequently used material and there needs to be assurance of the quality of
water used.
Product-contact utilities, such as compressed air and Nitrogen, must not be forgotten.
These will typically be filtered close to the point of use.
5.1.4 Drying:
If the water activity of the product is reduced below 0.6, then microbial growth will be
suppressed.

5.2. pH:

Values > 10 or < 2 generally being detrimental to micro-organisms

5.3. Osmolarity of solutions:

High osmolarity typically being detrimental to micro-organisms

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