12

Qualification of Water and Air

Handling Systems

Kunio Kawamura
Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan

I. PURPOSE OF VALIDATION

High-quality water and air are essential for the manufacture of pharmaceuticals.
Water is the most commonly used raw material in pharmaceutical manufactur-
ing; it is indirectly used in the manufacture of all dosage forms for cleaning
manufacturing equipment, and is also used as a major component which consti-
tutes injectable products. It is the one raw material that is usually processed by
the pharmaceutical manufacturer prior to use because it cannot be used as sup-
plied by the vendor. Water should be regarded as one of major raw materials for
the manufacture of pharmaceuticals whether or not it remains as a component of
the finished dosage form or is eliminated during the manufacturing process.
Water is thus an important raw material in GMP and in validating the manufac-
turing process.
Air supplied to the pharmaceutical manufacturing area or the air in the
environment of the pharmaceutical manufacturing area always contacts with
pharmaceuticals, and the quality of air influences the quality of the pharmaceuti-
cals manufactured, particularly in their cleanliness, particulates, and microbial
quality. Temperature and humidity in the manufacturing environment also influ-
ence the quality of the pharmaceuticals manufactured.
The importance of air quality and air handling system are described in
CFR 211-46 as part of GMP.
The USP identifies several grades of water that are acceptable for use in
pharmaceuticals, and also defines the quality of the environment or the quality
of air for the manufacturing of pharmaceuticals according to its criticality.

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 Water and the environment must be periodically monitored for these qual-
ity attributes, and in some instances the results are not available for days after
the sample is obtained. Meanwhile, the water would have been used to manufac-
ture a great number of pharmaceutical products or else the products would have
already been exposed to the environment. Water treatment and air handling
systems are highly dynamic, and careful attention has to be paid to their opera-
tion, even though this may sometimes be somewhat unreliable. Consequently,
they must be validated and then closely monitored and controlled.
Validation is defined as “a documented program that provides a high de-
gree of assurance that a specific process, method, or system will consistently
produce a result meeting pre-determined acceptance criteria” [1].
The purpose of validation is to demonstrate the capability of the water
treatment and air handling system to continuously supply the required quantity
of water and air with the specified quality attributes. “Documented” means to
provide documented “evidence.” Validation provides the system owner with the
means of assessing when a water treatment and/or air handling system is operat-
ing outside established control parameter limits and provides a means for bring-
ing the system back into a state of control. It results in written operating and
maintenance procedures for personnel to follow, which in turn helps ensure
consistent system performance.

II. VALIDATION STRATEGY

A. Validation Concept
The basic strategy is to prove the performance of processes or systems under
all conditions expected to be encountered during future operations. To prove the
performance, one must demonstrate (document) that the processes or systems
consistently produce the specified quantity and quality of water and/or air when
operated and maintained according to specific written operating and mainte-
nance procedures. In other words, validation involves proving
1. Engineering design
2. Operating procedures and acceptable ranges for control parameters
3. Maintenance procedures
To accomplish this, the system must be carefully designed, installed, and tested
during and after construction, and therefore for a prolonged period of time under
all operating conditions.
Variations in daily, weekly, and annual system usage patterns must be
validated. For example, water may be drawn from the system for manufacturing
use only during normal working hours; there may be no demands on the system
at other times during the 24-hr cycle. The system may be idle on weekends and

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 on holidays, which could extend for as long as 4 days or more. In addition,
many firms have annual plant maintenance shutdowns, typically in the summer,
and systems must be sanitized and restarted prior to use, and of course emer-
gency shutdowns can occur at any time and the system must be brought back
online. Systems with ion exchange resins (deionizers) must be at least partially
shut down to regenerate the resins when the chemical quality of the treated
water drops below a specified level. (This could be a matter of a few days or
even a few months, depending on the quantity of water processed through the
system and other factors.) For the air handling system, the same kinds of issues
exist. Clean rooms should be maintained at their required cleanliness level, even
during the time of no manufacturing operation. If the cleanliness is broken or
the air handling system stops, the whole clean area has to be made clean accord-
ing to the initial validation procedure and assessment. Water treatment and/or
air handling systems must be validated under all of these normal operating con-
ditions in order to prove the adequacy of the engineering design and the effec-
tiveness of the operating, control, and maintenance procedures.

B. Validation Life Cycle

1. Determination of Quality Attributes
In performing the validation, defining the quality attributes—that is, gaining a
clear understanding of the required quality and intended use—is the most impor-
tant issue, and should be determined before starting the validation. Without
defining required quality attributes we cannot establish validation protocols,
which are the basis of all validation studies.

2. The Validation Protocol

A validation protocol is defined as
A written plan stating how validation will be conducted and defining accep-
tance criteria. For example, the protocol for a manufacturing process identi-
fies process equipment, critical process parameters/operation ranges, prod-
uct characteristics, sampling, and test data to be collected, number of
validation runs, and acceptable test results [1].

The validation protocol is a detailed plan for conducting a validation

study. It is drafted by the individual or task group responsible for the project,
reviewed for content and completeness following the firm’s protocol review
procedure, and approved by designated individuals. It describes the responsibili-
ties of each individual or unit involved in the project.
All protocols, whether for IQ (installation qualification)/OQ (operational
qualification) of new equipment or for validating a new process, have the same

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 basic format. They start with an objective section, which describes the reasons
for conducting the validation study as well as the results to be achieved. Next
there is a scope section. Here what is to be included and excluded from the
study is specified, effectively establishing the boundaries for the study.
Following the objective and scope sections is a detailed description of the
process/equipment to be validated. Here block diagrams of equipment, batch
formula and master manufacturing records, process flow diagrams, and other
documents that will help with the descriptive process are essential and should
be attached to the protocol. The protocol should contain a detailed description
of the sampling and testing schedule and procedures and clearly state the accep-
tance criteria for each stage of validation, such as DQ (design qualification), IQ,
OQ, and PQ (performance qualification). The number of times that specific
trials will be replaced in order to demonstrate reproducibility of results must be
specified.
The protocol should be endorsed by designated representatives of each unit
that will participate in the validation study. This is an essential step for validation
study. It should be described that the protocol is accepted by responsible persons,
and that each unit understands and agrees to fulfill its responsibilities as stated in
the protocol. Subsequent changes to the protocol, should they be necessary, must
be endorsed by the same individuals. Protocol addenda are sometimes necessary
because circumstances later arise that were impossible to anticipate when the study
was planned and the protocol drafted. In addition to approvals, the validation
protocol should have the appended data sheets, which are to be filled with data
obtained from the validation studies and compared with the criteria.

3. Steps of Validation
Validation plans for water and air systems typically include the following steps
(Fig. 1):
1. Establishing standards for quality attributes of water and air to man-
ufacture pharmaceuticals.
2. Defining systems and subsystems suitable to produce the desired
water and air by considering the quality grades of water and air.
3. Designing equipment, controls, and monitoring technologies.
4. Establishing standards for operating parameters of the selected
equipment of the system.
5. Developing an IQ stage consisting of instrument calibrations, inspec-
tions to verify that the drawings accurately depict the as-built config-
uration of the system, and special tests to verify that the installation
meets the design requirements. These items include pipe and instru-
ment drawings, air pressure differentials, air velocities, and airflow
patterns.

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 Figure 1 Validation life cycle of water and air system. (From Ref. 2.)

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 6. Developing an OQ stage consisting of tests and inspections to verify
that the equipment, system alerts, and controls are operating.
7. Establishing alert and action levels for the operational standards and
routine control. This phase of qualification may overlap with aspects
of the next step.
8. Developing a prospective PQ stage to confirm the appropriateness of
critical process parameter operating ranges. System reproducibility is
to be demonstrated in this stage over an appropriate time period.
During this phase of validation, alert and action levels for key qual-
ity attributes of water, such as TOC, pH, particulates and microbes,
and operating parameters for an air system (e.g., temperature, time,
air pressure differential, airflow velocity, and air exchange rate) are
verified.
9. Supplementing a validation maintenance program (also called con-
tinuous validation life cycle) that includes a mechanism to control
changes to the system and establishes and carries out scheduled pre-
ventive maintenance, including recalibration of instruments.
10. Instituting a schedule for periodic review of the system performance
and requalification.
11. Completing protocols and documenting steps 1 through 10 [2].

4. Control During Routine Operation

Revalidation and Change Control. Once the validation is completed, the
standard operating procedures (SOPs) are formalized. Routine operation should
be performed according to the established SOP.
Any proposed changes should be evaluated for their impact on the whole
system. The necessity for requalifying the system because of changes should be
determined. Revalidation and evaluation should be performed depending upon
the impact that might be caused by the change.
Alert and Action Levels. Validated and established systems should be
periodically monitored to confirm that they continue to operate within their
design specifications and consistently produce water or air of acceptable quality.
Monitored data may be compared to established process parameters or product
specifications. A refinement to the use of process parameters and product speci-
fications is the establishment of alert and action levels, which signal a shift in
process performance. Alert and action levels are distinct from process parame-
ters and product specifications in that they are used for monitoring and control
rather than accept or reject decisions. The levels should be determined based on
the statistical analysis of the data obtained by monitoring at the PQ step.
Alert levels are levels or ranges that when exceeded indicate that a process
may have drifted from its normal operation condition. Alert levels indicate a

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 warning and do not necessarily require a corrective action. Exceeding an action
level indicates that corrective action should be taken to bring the process back
into its normal operating range [2,8].

III. QUALIFICATION OF WATER SYSTEMS

A. Required Quality for Water for
Pharmaceutical Purposes
Water is one of the most widely used substances, and raw materials, or an
ingredient in the production, processing, and formulation of pharmaceuticals.
Control of the organic and inorganic impurities and microbiological qual-
ity of water is important because proliferation of micro-organisms ubiquitous in
water may occur during the purification, storage, and distribution of this sub-
stance. Although there are various quality grades of water used for pharmaceuti-
cal purposes, all kinds of water are usually manufactured from drinking water
or comparable grade water as a source water.
Grades of water are closely related to the manufacturing methods and
distribution system of water. Major differences among these grades of water
consist of the following quality attributes:
Microbial counts
Endotoxin, which is due to the presence of microbes
Organic and inorganic impurities
Grades of water specified in the compendia (USP) are classified according
to the above quality attributes as
1. Potable water
2. Purified water
3. Water for injection
4. Sterile water for injection
5. Sterile water for inhalation
6. Sterile water for irrigation
7. Sterile bacteriostatic water for injection
Grades of water specified in the Pharmacopeia (USP) are summarized in
Table 1. “Water for injection” (WFI) is the most purified water, and careful
attention should be paid to the validation of its manufacturing process.

B. Selection of Water for Pharmaceutical Purposes

The quality attributes of water for a particular application are dictated by the
requirement of its usage. Sequential steps that are used for treating water for
different pharmaceutical purposes are shown in Figure 2 [6].

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 Table 1 Specifications of Water for Pharmaceutical Use

Microbial Particulate
Inorganics control Microbial limit matter Endotoxin

Source water (I) −a −a −a −a −a

City water (potable) (II) Reg.b Reg.b Reg.b −a −a
Purified water (deionized) (III) +c +c 100 CFU/mLd −a −a
Purified water (membrane) (IV) +c +c 100 CFU/mLd −a +
WFI (rinse) (V) +c +c 100 CFU/mLd −a +
WFI (preparation) (VI) +c +c 100 CFU/mLd +f <0.25 EU/mle
WFI (LVP) (VII) +c +c 100 CFU/mLd +f <0.25 EU/mle
a
No control, no specifications.
b
City water regulations.
c
Controlled to be less than city water specifications. Microbial counts in deionized water should be carefully controlled.
d
According to the specifications by USP/EP, and recommended criteria final rinse water: 10 CFU/100 mL.
e
Cooling water used for sterile products: 1 CFU/100 mL.
f
Particulate matter for LVP: (>10 µm, 20/ml), >25 µm, 2/ml).

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 Figure 2 Water for pharmaceutical purposes. (From Ref. 2.)

The manufacturing method and distribution system also have a close rela-
tion with the construction design of facilities and equipment.
1. Selection of the most suitable quality grade of water for its intended use.
2. Determination of the water manufacturing system elements, including
facility and equipment.
3. Design of water manufacturing system, including the design of system
equipment.
4. After construction of the water system is completed based on its de-
sign, the system has to be scrutinized as to whether it has been built
to design specification or not.

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 5. After confirming the installation of facility and equipment, the quality
of water produced is examined from various viewpoints according to
the predetermined specifications.
6. In the routine production of water, representative quality items of wa-
ter have to be monitored to confirm the performance of normal opera-
tion, and if any undesirable trends or out of specification values are
found, corrective action should be taken.
The steps of checking design and construction, confirming proper installa-
tion and operation, and documenting these processes are collectively called qual-
ification or validation. In case of any system change or changes to equipment,
the same kinds of procedures should be implemented.
Water for pharmaceutical purposes, and selection of water grade for phar-
maceuticals are summarized in Figures 1 and 2. Specifications of various kinds
of water are summarized in Table 1.
Major items of quality attributes that should be controlled and specified
for pharmaceutical use are
1. Organic impurities
2. Inorganic impurities
3. Particulates
4. Microbes
5. Endotoxin

C. Design Qualification of Water Systems

The quality attributes of water for a particular application are dictated by the
requirements of its usage. Production of pharmaceutical water employs a combi-
nation of sequential unit operations (processing steps) that address specific water
quality attributes.
The validation plan should be designed to establish the suitability of the
system and provide a thorough understanding of the purification mechanism,
range of operating conditions, required pretreatment, and the most likely mode
of failure. It is also necessary to demonstrate the effectiveness of the monitoring
scheme and to establish the requirements for validation maintenance. The selec-
tion of specific unit operations and design characteristics for a water system
should take into consideration the quality of the feed water, the technology
chosen for subsequent processing steps, the extent and complexity of the water
distribution system, and the appropriate requirements. In a system for WFI, the
final process (distillation, reverse osmosis, or ultrafiltration) must have effective
bacterial endotoxin reduction capability and must be validated for each specific
equipment unit. The final unit operations used to produce WFI have been lim-
ited to distillation, reverse osmosis, and/or ultrafiltration. Distillation has a long

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 history of reliable performance for the production of WFI. Other technologies,
such as reverse osmosis and ultrafiltration, may be suitable in the production of
WFI if they are appropriately validated for each specific set of equipment.
Typical sequential processing steps that are used for manufacturing puri-
fied water (PW) are shown in Figure 3. A distilled water distribution system
that is commonly used for WFI is shown in Figure 4.
1. Step 1 is the combination of prefilter, carbon filter, and ion exchanger
(softener). After chlorine is removed, attention has to be paid to pre-

Figure 3 Typical sequential processing steps for water treatment.

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 Figure 4 Typical sequential processing steps for distilled water treatment. When cool-
ing water is not needed, water will circulate at 80°C or above. During use the cooling
water valve is open and the loop outlet valve is closed. After use the loop outlet valve
is kept closed and the loop drain valve opened to flush all the cooled water to drain.

vent microbial growth. For this purpose, ultraviolet light is installed

and the water is circulated.
2. Step 2 is a reverse osmosis process, after adjusting temperatures by
heat exchanger.
After the reverse osmosis process, inorganic impurities are completely
removed by anion exchanger and cation exchanger. An ultraviolet light is in-
stalled and the water is circulated to prevent microbial growth.
The obtained water can be used for nonparenteral dosage forms. For the
parenteral purpose, water obtained in this way is usually distilled.
Water for injection obtained by distillation is circulated through the main
loop and subloop at 80. During use the cooling water valve is open and the loop
outlet valve is closed. After use the loop outlet valve is kept closed and the loop
drain valve is opened to flush all the cooled water to drain.
A typical evaluation process to select an appropriate water quality for a
particular pharmaceutical purpose is shown in the decision tree in Figure 5 [2].

D. Qualification of Equipment and Components

for Water System
Equipment and components used for the water system must maintain sanitary
integrity and be anticorrosive and assured for technical integrity.

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 Figure 5 Selection of water for pharmaceutical purpose. *, Water for sterile BPCs or
dosage forms must be rendered sterile if there is not a sterilization step following addi-
tion; ‡, microorganism control can occur either in water treatment or in BPC process; †,
endotoxin removal can occur either in water treatment or in BPC process; §, NPDWR
water—water meeting EPA national primary drinking water regulations. (From Ref. 2.)

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 1. Components
Selection should be made with assurance that it does not create a source for
contamination intrusion.

2. Piping and Installation

Stainless steel welds should provide reliable joints that are internally smooth
and corrosion-free. Low carbon stainless steel (SUS 304L, 316, and 316L) com-
patible wire filler, and where necessary, inert gas, automatic welding machines,
and regular inspection and documentation, help to ensure acceptable weld qual-
ity. Follow-up cleaning and passivation are important for removing contamina-
tion and corrosion products and to re-establish the passive corrosion-resistant
surface. Piping systems should be installed and sloped in such a way that they
are completely self-draining. Complete drainage is important, as it prevents the
formation of standing “pools” of liquid that can support the growth of microbes.
Further, properly sloped piping prevents the formation of condensate “plugs”
that can cause cold spots during SIP, and most important it allows for the free
drainage of all rinsing and washing solutions during CIP, which enhances clean-
ing efficiency. The slope is normally 1/200 to 1/100. Isometric drawings of
piping systems for water systems are essential for the design and installation
qualification of both water supply and CIP/SIP piping systems.
3. Material
Materials of construction should be selected to be compatible with mater-
ials used as control measures, such as sanitizing, cleaning, and passivating
media.
Plastic materials can be fused (welded) in some cases and also require
smooth, uniform internal surfaces. Adhesives should be avoided due to the po-
tential for voids and chemical reactions. Mechanical methods of joining, such
as flange fittings, require care to avoid the creation of offsets, gaps, penetrations,
and voids. Control measures include good alignment, properly sized gaskets,
appropriate spacing, uniform sealing force, and the avoidance of threaded fit-
tings.
Temperature rating is a critical factor in choosing appropriate materials
because surfaces may be required to handle elevated operating and sanitiza-
tion temperatures. Should chemicals or additives be used to clean, control, or
sanitize the system, materials resistant to these chemicals or additives must be
utilized.
4. Surface Polishing
The finish on metallic materials such as stainless steel, whether it be a refined
mill finish, polished to a specific grit, or an electropolished treatment, should

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 complement system design and provide satisfactory corrosion and microbial ac-
tivity resistance.

5. Dead Legs
Dead legs pose two problems for CIP. First, cleaning fluids must be able to
flush out trapped gas pockets in order to wet all the piping surfaces in the dead
legs. Second, fresh cleaning fluid must flush the dead leg to maintain rapid
cleaning rates. Dead legs should not be greater in length than six diameters (6D)
of the unused portion measured from the axis of the pipe in use.

6. Valves
The most commonly used valves in process piping systems for PW and WFI
used for pharmaceutical manufacturing are diaphragm valves. This is because
they are easily CIP-cleanable and provide complete containment of in-process
materials. Diaphragm valves are limited in the ways they may be installed for
free drainage; they sometimes are prone to leakage and they have a relatively
high pressure drop as compared with other types of valves.
For situations in which complete containment is not required, “plunger-
type” compression valves of hygienic design may be used. These have several
advantages over diaphragm valves regarding installation and operation but they
do not provide complete containment.
Ball and butterfly valves are also commonly used in water treatment sys-
tems. Diaphragm valves should be used downstream from the unit that removes
dissolved solids (reverse osmosis unit or deionizer), however, because of their
inherent ease of sanitation.

7. Pumps
Pumps should be of sanitary design with seals that prevent contamination of the
water. Pumps moving water for manufacturing or final rinsing, water for cooling
the drug product after sterilization, and in-process or drug product solutions
shall be designed to utilize water for injection as a lubricant for the seals. Sev-
eral types of CIP-cleanable pumps are commonly used in water systems or phar-
maceutical manufacturing processes. These include centrifugal, rotary lobe, peri-
staltic, and diaphragm pumps, of which all but the centrifugal pump provide
positive displacement.

8. In-Line Instrumentation
In-line instruments or sensors are necessary components for automated pro-
cesses. For ease of cleaning, sensors should be chosen that directly mount onto
vessel nozzles or piping tees with minimum dead leg distances. Also, the instru-

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 ments should be of a cleanable design and constructed to similar standards as
those for process equipment and piping.

9. Pressure Gauges
Sanitary diaphragm-style pressure gauges should be used when possible, as they
are very cleanable. When pressure gauges are installed in process piping, the
diameter should be less than 6D.

10. Heat Exchangers

Heat exchangers should be double tube sheet or concentric tube design. Heat
exchangers other than the welded double-concentric tube type or double-tube
sheet type must employ a pressure differential and a means for monitoring the
differential. The pressure differential shall be such that the fluid requiring a
higher microbial quality shall be that with the greater pressure.

11. Distillation
Distillation equipment is used to produce USP WFI-quality water. The distilla-
tion process removes dissolved solids not otherwise removed by deionizers or
RO units.
The chemical quality of the steam supplied to the still must be controlled
to prevent recontamination of the distillate. Also, the condenser must be of a
double-tube design to prevent condenser coolant from coming into direct contact
with the distallate, thereby causing recontamination.
Separation of mists by the distillation process is important to remove the
endotoxin or other contaminant. Distillators have their own upper limits of
throughput capacity. Usually the more the amount of water generated at the unit
period, the more the conductivity increases at the range of maximum capacity.
This means that the purity of the water becomes worse.

12. Filters
Final filters of water for injectable products may not be used at any point in the
piping system of water for manufacturing or final rinse.
Water storage tank vent filters must be equipped with a sterilizing air filter
in order to prevent the air, which displaces water drawn from the tank, from
microbiologically contaminating the water. The filter must be hydrophobic in
order to prevent condensation from blinding the filter and preventing air entry
or escape from the tank. It also must have a mean porosity of less than 1 µm.
Water filters are used in various locations in water treatment systems for
two basic purposes: removal of undissolved solids, some of which are added to
the water by various components of the water treatment system, and removal of

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 bacterial contaminants. Filters are commonly used downstream from carbon
beds and resin beds and on the incoming water supply line, and they are typi-
cally in the order of 10 to 15-µm mean porosity. Membrane filters of 0.2 µm
are used to remove bacteria. Filters must be properly maintained in order to
keep the water treatment system operating efficiently and to prevent them from
becoming a source of bacterial and endotoxin contamination.
Microbes are not destroyed by bacteria-retentive filters but instead become
concentrated in and on them. Certain bacteria have the capability of growing
through a membrane filter. Also, filters can become damaged by frequent or
sudden changes in water pressure (water hammer).
13. Deionizers
These devices are used to remove dissolved solids from the feed water. Dioniz-
ers use ion exchange resins to remove charged particles. Cation resin beds re-
move negatively charged particles; anion resins remove positively charged parti-
cles. Mixed bed deionizers (containing both cation and anion exchange resins)
are commonly used to give the water a final “polishing” treatment. Resins lose
their ability to remove charged particles and must be periodically regenerated
using strong caustic and acid solutions. This treatment also sanitizes the resin
beds, which, like carbon beds, are a fertile breeding ground for bacteria when
improperly maintained.
14. Auxiliary Equipment
1. Backflow of liquids shall be prevented at points of interconnection of
different systems.
2. Pipelines for the transmission of water for manufacturing or final
rinse and other liquid components shall be constructed of welded
stainless steel (nonrusting grade) equipped for sterilization with
steam, except that sanitary stainless steel lines with fittings capable
of disassembly may be immediately adjacent to the equipment or
valves that must be removed from the lines for servicing and replace-
ment.
3. Auxiliary equipment and fittings that require seals, gaskets, dia-
phragms, filter media, and membranes should exclude materials that
permit the possibility of extractables entry, shedding, and microbial
activity [7–12].
15. Ultraviolet Light
Ultraviolet light (UV) is not effective enough to eliminate existing biofilm.
When coupled with conventional thermal or chemical sanitization technologies,
however, it is most effective and can prolong the interval between system sani-
tizations.

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 The use of UV light also facilitates the degradation of hydrogen peroxide
and ozone. The most effective biocidal wavelength is 253.7 nm. The amount of
light at 255 nm emitted by a UV light decreases with time, so lamps have to be
monitored and replaced when necessary.

16. Wastewater
Waste liquids shall be introduced to sewers through trapped drains. Drains from
equipment shall be designed with an atmospheric break to prevent back-
siphonage.
All stills and tanks holding liquid requiring microbial control shall have
air vents with non-fiber-releasing sterilizable filters capable of preventing micro-
bial contamination of the contents. Such filters shall be designed and installed
so that they do not become wet. Filters shall be sterilized and installed aseptic-
ally. Tanks for holding water require air vents with filters [7,10].

E. Sanitization
Microbial control in water systems is achieved primarily through sanitization
practices. Systems can be sanitized using either thermal or chemical means. In-
line UV light can also be used to “sanitize” water in the system continuously.

1. Thermal Approaches
Thermal approaches to system sanitization include periodic or continuously cir-
culating hot water and the use of steam. These techniques are limited to systems
that are compatible with the higher temperatures needed to achieve sanitization,
such as stainless steel and some polymer formulations. Hot water circulation is
effective or essential for this purpose, especially for the WFI system.

2. Chemical Methods
Chemical methods, where compatible, can be used on a wider variety of con-
struction materials. These methods typically employ oxidizing agents such as
hypochlorite, hydrogen peroxide, ozone, or per-acetic acid. Hypochlorites are
effective sanitizers but are difficult to flush from the system and tend to leave
biofilms intact.

3. Validation of Sanitization Steps

Sanitization steps require validation to demonstrate the capability of reducing
and holding microbial contamination at acceptable levels. Validation of thermal
methods should include a heat distribution study to demonstrate that sanitization
temperatures are achieved throughout the system. Validation of chemical meth-

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 ods requires a demonstration of adequate chemical concentrations throughout
the system. In addition, when the sanitization process is completed, effective
removal of chemical residues must be demonstrated.
The frequency of sanitization is generally dictated by the results of system
monitoring. Conclusions derived from the trend analysis of the microbiological
data should be used as the alert mechanism for maintenance. The frequency
of sanitization should be established so that the system operates in a state of
microbiological control and does not exceed alert levels.

4. SIP/CIP
For a WFI or highly purified water system, the SIP/CIP method is usually ap-
plied. In an SIP/CIP system, sterilization and/or cleaning take in place in situ or
without dissembling the equipment. Consequently, the result of sterilization and/
or cleaning is confirmed by the process parameters previously validated. Based
on process parameters and their ranges previously determined by the process
validation, the SIP/CIP process can be confirmed whether or not it is completely
sterilized and cleaned. This is a typical application of the concept of validation.

5. Hot Water Circulation of WFI System

Hot water circulation systems circulate hot water through all pipelines from the
storage tank through all use points to return to the storage tank. By a combina-
tion of hot water circulation system and SIP, microbial quality of WFI can be
guaranteed to be 0 cfu per 100 ml. Once the water in the system is drained out,
the entire system must be steam-sterilized. By applying hot water circulation
and SIP, formation of any biofilm can be prevented if the piping and installation
are well designed and there are no dead legs [2,7–9,11,12].

F. Sampling Considerations
Water systems should be monitored at a frequency that is sufficient to ensure
that the system is in control and continues to produce water of acceptable qual-
ity. Samples should be taken from representative locations within the processing
and distribution system. Established sampling frequencies should be based on
system validation data and should cover critical areas. Unit operation sites might
be sampled less frequently than point-of-use sites. The sampling plan should
take into consideration the desired attributes of the water being sampled. Be-
cause of their more critical microbiological requirements, systems for WFI may
require a more rigorous sampling frequency.
When sampling water systems, special care should be taken to ensure that
the sample is representative. Sampling ports should be sanitized and thoroughly
flushed before a sample is taken. Samples for microbiological analysis should

Copyright © 2003 Marcel Dekker, Inc.

 be tested immediately or suitably protected to preserve the sample until analysis
can begin. The sampling operation itself might often cause a microbial contami-
nation.

1. Biofilm, Planktonic Micro-Organisms, and Benthic

Micro-Organisms
Samples of flowing water are only indicative of the concentration for planktonic
(free-floating) micro-organisms present in the system. The number of microbes
determined by the water system monitoring is an indication of floating microbes
in water; that is, planktonic micro-organisms. Benthic (attached) micro-organ-
isms in the form of biofilms are generally present in greater numbers and are
the source of the planktonic population.
The major purpose of monitoring microbes is to identify the generation of
biofilms and to find the locations of biofilms, if any. The purpose of sanitization
is to kill and destroy the biofilm after detecting the location of the biofilms. The
planktonic population, whose number of micro-organisms in water is monitored,
should be understood and utilized to indicate biofilms in the system. The num-
ber of microbes in water is an indicator of system contamination levels and is
the basis for the system alert levels.
If there were no microbials in water, there would not be any biofilms in
the system. If any microbials are detected in the system, there must be biofilms
in some locations. Biofilm is formed in stagnant places, such as valves, dead
ends, or unsuitably sloped piping. Detecting micro-organisms and biofilms is
one method of controlling the cleanliness of the system. The other method is to
completely eliminate the stagnant places or dead ends that might cause biofilms.
From such viewpoints, the design and construction of a desirable water system
as described in sec. III.D is the fundamental way to prevent the formation of
biofilms, and consequently to both reduce the number of micro-organisms and
prevent the generation of micro-organisms in the system.

2. Operation, Monitoring, and Control

Monitoring programs should be established to ensure that the water system re-
mains in a state of control. The program should include
1. Procedures for operating the system
2. Monitoring programs for critical quality attributes and operating con-
ditions, including calibration of critical instruments
3. Schedule for periodic sanitization
4. Preventive maintenance of components
5. Control of changes to the mechanical system and to operating condi-
tions

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 3. Operating Procedures
Procedures for operating the water system and performing a routine monitoring
program should be established based on the validation study. The procedures
should be well documented, detail the function of each job, assign who is re-
sponsible for performing the work, and describe how the job is to be conducted.

4. Monitoring Program
Critical quality attributes and operating parameters should be documented and
monitored. The program may include a combination of in-line sensors or record-
ers (e.g., a conductivity meter and recorder), manual documentation of opera-
tional parameters (such as carbon filter pressure drop), and laboratory tests (e.g.,
total microbial counts). The frequency of sampling, the requirement for evaluat-
ing test results, and the necessity for initiating corrective action should be in-
cluded.

G. Microbial Considerations
The major exogenous source of microbial contamination is source or feed water.
At a minimum, feed water quality must meet the quality attributes of potable
water for which the level of coliforms is regulated. A wide variety of other
micro-organisms, chiefly gram-negative bacteria, may be present. These micro-
organisms may compromise subsequent purification steps. Examples of other
potential exogenous sources of microbial contamination include unprotected
vents, faulty air filters, backflow from contaminated outlets, drain air breaks,
and replacement activated carbon and deionizer resins. Sufficient care should
be given to system design and maintenance in order to minimize microbial con-
tamination from these sources.
Micro-organisms present in feed water may adsorb to carbon beds, deion-
izer resins, filter membranes, and other unit operation surfaces and initiate the
formation of a biofilm [2,8].

H. Endotoxin
Endotoxins are lipopolysaccharides from the cell envelope that is external to the
cell wall of gram-negative bacteria. Gram-negative bacteria readily form biofilm
that can become a source of endotoxin. Endotoxin may be associated with living
micro-organisms or fragments of dead micro-organisms, or may be free molecules.
The free form of endotoxin may be released from cell surfaces or biofilm that
colonize the water system, or they may enter the water system via the feed water.
Endotoxin should be eliminated by means of distillation, reverse osmosis,
and/or ultrafiltration. Generation of endotoxin is prevented by controlling the

Copyright © 2003 Marcel Dekker, Inc.

 introduction of micro-organisms and microbial proliferation in the system. This
may be accomplished through sanitization and sterilization.
The presence of endotoxin should be monitored by LAL method in the
routine operation. Endotoxin can be removed by means of distillation, reverse
osmosis, and/or ultrafiltration. Incomplete separation of mist in distillation, how-
ever, and leakage in membrane of reverse osmosis or ultrafiltration cause con-
tamination with endotoxin. After these separation processes, of course, contami-
nation of microbes or growth of microbes causes endotoxin contamination [2,8].

I. Methodological Considerations
The objective of a water system microbiological monitoring program is to pro-
vide sufficient information to control the microbiological quality of the water
produced. An appropriate level of control may be maintained by using data
trending techniques and limiting specific contraindicated micro-organisms, con-
sequently it may not be necessary to detect all of the micro-organisms present.
The methods selected should be capable of isolating the numbers and types of
organisms that have been deemed significant relative to system control and
product impact for each individual system [2,8].

J. Continuous Automatic Monitoring of Water

Monitoring and feeding back the data are important in maintaining the perfor-
mance of water systems. By applying an automatic continuous monitoring sys-
tem combined with the method of trend analysis, processes can be maintained
in a much more stable state. For example, this can be achieved by applying
automatic continuous monitoring of TOC and conductivity of the water system.
TOC and conductivity are the major quality attributes of water by which
organic and inorganic impurities can be determined.

IV. QUALIFICATION OF AIR HANDLING SYSTEMS

A. Purposes of an Air Handling System
The purposes of an air handling system are
1. To prevent microbial contamination of sterile products and of clean
areas
2. To prevent the spreading and contamination of virus, pathogenic, and
spore-forming microbes used in the manufacturing of pharmaceuticals
3. To prevent spread and contamination of penicillin or other sensitizing
drugs, cytotoxic drugs, and drugs with strong pharmacological action

Copyright © 2003 Marcel Dekker, Inc.