1231WATER FOR PHARMACEUTICAL PURPOSES, chemical structure, molecular formula, Reference Standards

á1231ñWATER FOR PHARMACEUTICAL PURPOSES

Water is the most widely used substance,raw material,or ingredient in the production,processing,and formulation of compendial articles.Control of the microbiological quality of these waters is important because proliferation of microorganisms ubiquitous to water may occur during the purification,storage,and distribution of this substance.If water is used in the final product,these microorganisms or their metabolic products may eventually cause adverse consequences.

Water that is used in the early stages of the production of drug substances and that is the source or feed water for the preparation of the various types of purified waters must meet the requirements of the National Primary Drinking Water Regulations (NPDWR)(40CFR141)issued by the Environmental Protection Agency (EPA).Comparable regulations for drinking water of the European Union or Japan are acceptable.These requirements ensure the absence of coliforms,which,if determined to be of fecal origin,may portend or indicate the presence of other microorganisms of fecal origin,including viruses that may be pathogenic for humans.On the other hand,meeting these National Primary Drinking Water Regulations would not rule out the presence of other microorganisms,which,while not considered a major public health concern,could,if present,constitute a hazard or be considered undesirable in a drug substance or formulated product.For this reason,there are many different grades of pharmaceutical waters.

TYPES OF WATER

Drinking Water— Drinking Water is not covered by a compendial monograph but must comply with the quality attributes of the EPA NPDWRor comparable regulations of the European Union or Japan.It may be derived from a variety of sources,including a public water utility,a private water supply (e.g.,a well),or a combination of more than one of these sources.Drinking Water may be used in the early stages of chemical synthesis and in the early stages of the cleaning of pharmaceutical manufacturing equipment.It is the prescribed source feed water for the production of pharmaceutical waters.As seasonal variations in the quality attributes of the drinking water supply can occur,processing steps in the production of pharmaceutical waters must be designed for this characteristic.

Purified Water— Purified Water (see USPmonograph)is used as an excipient in the production of official preparations;in pharmaceutical applications,such as cleaning of certain equipment;and in the preparation of some bulk pharmaceutical chemicals.Purified Water must meet the requirements for ionic and organic chemical purity and must be protected from microbial proliferation.It is prepared using Drinking Water as a feed water and is purified using unit operations that include deionization,distillation,ion exchange,reverse osmosis,filtration,or other suitable procedures.Purified Water systems must be validated.

Purified Water systems that produce,store,and circulate water under ambient conditions are susceptible to the establishment of tenacious biofilms of microorganisms,which can be the source of undesirable levels of viable microorganisms or endotoxins in the effluent water.These systems require frequent sanitization and microbiological monitoring to ensure water of appropriate microbiological quality at the points of use.

Sterile Purified Water— Sterile Purified Water is Purified Water that is packaged and rendered sterile.It is used in the preparation of nonparenteral compendial dosage forms where a sterile form of Purified Water is required.

Water for Injection— Water for Injection (see USPmonograph)is an excipient in the production of injections and for use in pharmaceutical applications,such as cleaning of certain equipment and preparation of some bulk pharmaceutical chemicals.The source or feed water for this article is Drinking Water,which may have been preliminarily purified but which is finally subjected to distillation or reverse osmosis.It must meet all the chemical requirements for Purified Water and in addition the requirements under Bacterial Endotoxins Test á85ñ.It also must be protected from microbial contamination.The system used to produce,store,and distribute Water for Injection must be designed to prevent microbial contamination and the formation of microbial endotoxins,and it must be validated.

Sterile Water for Injection— Sterile Water for Injection (see USPmonograph)is Water for Injection that is packaged and rendered sterile.Sterile Water for Injection is intended for extemporaneous prescription compounding and is distributed in sterile units.It is used as a diluent for parenteral products.It is packaged in single-dose containers not larger than 1Lin size.

Bacteriostatic Water for Injection— Bacteriostatic Water for Injection (see USPmonograph)is sterile Water for Injection to which has been added one or more suitable antimicrobial preservatives.It is intended to be used as a diluent in the preparation of parenteral products.It may be packaged in single-dose or multiple-dose containers not larger than 30mL.

Sterile Water for Irrigation— Sterile Water for Irrigation (see USPmonograph)is Water for Injection,packaged in single-dose containers of larger than 1L,that is intended to be delivered rapidly and is rendered sterile.It need not meet the requirement for small-volume injections under Particulate Matter á788ñ.

Sterile Water for Inhalation— Sterile Water for Inhalation (see USPmonograph)is Water for Injection that is packaged and rendered sterile and is intended for use in inhalators and in the preparation of inhalation solutions.

VALIDATION AND QUALIFICATION OF WATER PURIFICATION,STORAGE,AND DISTRIBUTION SYSTEMS

Establishing the dependability of pharmaceutical water purification,storage,and distribution systems requires an appropriate period of monitoring and observation.Ordinarily,few problems are encountered in maintaining the chemical purity of Purified Water and Water for Injection.However,it is more difficult to meet established microbiological quality criteria consistently.Atypical program involves intensive daily sampling and testing of major process points for at least one month after operational criteria have been established for each sampling point.

Validation is the procedure for acquiring and documenting substantiation to a high level of assurance that a specific process will consistently produce a product conforming to an established set of quality attributes.The validation defines the critical process parameters and their operating ranges.Avalidation program qualifies the design,installation,operation,and performance of equipment.It begins when the system is defined and moves through several stages:qualification of the installation (IQ),operational qualification (OQ),and performance qualification (PQ).Agraphical representation of a typical water system validation life cycle is shown in Fig.1.

Fig.1.Water System Validation Life Cycle

Avalidation plan for a water system typically includes the following steps:

Establishing standards for quality attributes and operating parameters.
Defining systems and subsystems suitable to produce the desired quality attributes from the available source water.
Selecting equipment,controls,and monitoring technologies.
Developing an IQstage consisting of instrument calibrations,inspections to verify that the drawings accurately depict the as-built configuration of the water system,and,where necessary,special tests to verify that the installation meets the design requirements.
Developing an OQstage consisting of tests and inspections to verify that the equipment,system alerts,and controls are operating reliably and that appropriate Alert and Action Levels are established.This phase of qualification may overlap with aspects of the next step.
Developing a prospective PQstage to confirm the appropriateness of critical process parameter operating ranges.Aconcurrent or retrospective PQis performed to demonstrate system reproducibility over an appropriate time period.During this phase of validation,Alert and Action Levels for key quality attributes and operating parameters are verified.
Supplementing a validation maintenance program (also called continuous validation life cycle)that includes a mechanism to control changes to the water system and establishes and carries out scheduled preventive maintenance,including recalibration of instruments.In addition,validation maintenance includes a monitoring program for critical process parameters and a corrective action program.
Instituting a schedule for periodic review of the system performance and requalification.
Completing protocols and documenting Steps 1–8.

PHARMACEUTICAL WATER SYSTEMS

The quality attributes of water for a particular application are dictated by the requirements of its usage.Sequential processing steps followed for treating water for different pharmaceutical purposes are shown in Fig.2.

Fig.2.Water for pharmaceutical purposes.

Atypical evaluation process to select an appropriate water quality for a particular pharmaceutical purpose is shown in the decision tree in Fig.3.

Fig.3.Selection of water for pharmaceutical purposes.

These diagrams may be used to assist in defining requirements for specific water uses and in the selection of unit operations.

PURIFIED WATER AND WATER FOR INJECTION SYSTEMS

The design,installation,and operation of systems to produce Purified Waterand Water for Injectioninclude similar components,control techniques,and procedures.The quality attributes of both waters differ only in the presence of a bacterial endotoxin requirement for Water for Injectionand in their methods of preparation,at least at the last stage of preparation.The similarities in the quality attributes provide considerable common ground in the design of water systems to meet either requirement.The critical difference is the degree of control of the system and the final purification steps needed to ensure bacterial and bacterial endotoxin removal.

Production of pharmaceutical water employs sequential unit operations (processing steps)that address specific water quality attributes and protect the operation of subsequent treatment steps.The final unit operations used to produce Water for Injectionhave been limited to distillation and reverse osmosis.Distillation has a long history of reliable performance and can be validated as a unit operation for the production of Water for Injection.Other technologies such as ultrafiltration may be suitable in the production of Water for Injection,but at this time,experience with this process is not widespread.

The validation plan should be designed to establish the suitability of the system and to provide a thorough understanding of the purification mechanism,range of operating conditions,required pretreatment,and 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.

Trials conducted in a pilot installation can be valuable in defining the operating parameters and the expected water quality and in identifying failure modes.However,qualification of the specific unit operation can be performed only as part of the validation of the installed operational system.

The selection 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 compendial requirements.For example,in the design of a system for Water for Injection,the final process (distillation or reverse osmosis)must have effective capability for bacterial endotoxin reduction and must be validated.

The following is a brief description of selected unit operations and the operation and validation concerns associated with them.This review is not comprehensive in that not all unit operations are discussed,nor are all potential problems addressed.The purpose is to highlight issues that focus on the design,installation,operation,maintenance,and monitoring parameters that facilitate water system validation.

Filtration technology plays an important role in water systems,and filtration units are available in a wide range of designs and for various applications.Removal efficiencies differ significantly from coarse filters,such as granular anthracite,quartz,or sand for larger water systems and depth cartridges for smaller water systems,to membrane filters for very small particle control.Unit and system configurations vary widely in type of filtering media and location in the process.(Use of membrane filters is discussed in a later paragraph.)

Granular or cartridge filters are used for prefiltration.They remove solid contaminants from the water supply and protect downstream system components from contamination that can inhibit equipment performance and shorten its effective life.Design and operational issues that may affect performance of depth filters include channeling of the filtering media,blockage from silt,microbial growth,and filtering-media loss.Control measures include pressure and flow monitoring,backwashing,sanitizing,and replacing filtering media.An important design concern is sizing of the filter to prevent channeling or media loss resulting from inappropriate water flow rates.

Activated carbon beds adsorb low-molecular-weight organic material and oxidizing additives,such as chlorine compounds,and remove them from the water.They are used to achieve certain quality attributes and to protect against reaction with downstream stainless steel surfaces,resins,and membranes.The chief operating concerns regarding activated carbon beds include the propensity to support bacteria growth;the potential for hydraulic channeling;the inability to be regenerated in situ,and the shedding of bacteria,endotoxins,organic chemicals,and fine carbon particles.Control measures include appropriate high water flow rates,sanitization with hot water or steam,backwashing,testing for adsorption capacity,and frequent replacement of the carbon bed.Alternative technologies such as chemical additives and regenerable organic scavenging devices can be used in place of activated carbon beds.

Chemical additives are used in water systems to control microorganisms by use of chlorine compounds and ozone,to enhance the removal of suspended solids by use of flocculating agents,to remove chlorine compounds,to adjust pH,and to remove carbonate compounds.Subsequent processing steps are required in order to remove the added chemicals.Control of additives and subsequent monitoring to ensure removal of additives and of any of their reaction products should be designed into the system and included in the monitoring program.

Organic scavenging devices use macroreticular anion-exchange resins capable of removing organic material and endotoxins from the water.They can be regenerated with appropriate biocidal caustic solutions.Operating concerns are associated with scavenging capacity and shedding of resin fragments.Control measures include testing of effluent,monitoring performance,and using downstream filters to remove resin fines.

Water softeners remove cations such as calcium and magnesium that interfere with the performance of downstream processing equipment such as reverse osmosis membranes,deionization columns,and distillation units.Water softener resin beds are regenerated with sodium chloride solution (brine).Concerns include microorganism proliferation,channeling due to inappropriate water flow rates,organic fouling of resin,fracture of the resin beads,and contamination from the brine solution used for regeneration.Control measures include recirculation of water during periods of low water use,periodic sanitization of the resin and brine system,use of microbial control devices (e.g.,UVand chlorine),appropriate regeneration frequency,effluent monitoring (hardness),and downstream filtration to remove resin fines.

Deionization (DI),electrodeionization (EDI)and Electrodialysis (EDR) are effective methods of improving the chemical quality attributes of water by removing cations and anions.

DIsystems have charged resins that require periodic regeneration with an acid and base.Typically,cationic resins are regenerated with either hydrochloric or sulfuric acid,which replace the captured positive ions with hydrogen ions.Anionic resins are regenerated with sodium or potassium hydroxide,which replace captured negative ions with hydroxide ions.Both regenerant chemicals are biocidal and offer a measure of microbial control.The system can be designed so that the cation and anion resins are separated or so that they form a mixed bed.Rechargeable resin canisters can also be used for this purpose.

The EDIsystem uses a combination of mixed resin,selectively permeable membranes,and an electric charge to provide continuous flow (product and waste concentrate)and continuous regeneration.Water enters both the resin section and the waste (concentrate)section.As it passes through the resin,it is deionized to become product water.The resin acts as a conductor enabling the electrical potential to drive the captured cations and anions through the resin and appropriate membranes for concentration and removal in the waste water stream.The electrical potential also separates the water in the resin (product)section into hydrogen and hydroxide ions.This separation permits continuous regeneration of the resin without the need for regenerant additives.

Electrodialysis (EDR)is a similar process that uses only electricity and selectively permeable membranes to separate,concentrate,and flush the removed ions from the water stream.The process,however,is less efficient than EDIbecause it contains no resin to enhance ion removal and current flow.Also,EDRunits require periodic polarity reversal and flushing to maintain operating performance.

Concerns for all forms of deionization units include microbial and endotoxin control;chemical additive effect on resins and membranes;and loss,degradation,and fouling of resin.Issues of concern specific to DIunits include regeneration frequency,channeling,complete resin separation for mixed bed regeneration,and mixing air contamination (mixed beds).Control measures vary but typically include recirculation loops,microbial control by UVlight,conductivity monitoring,resin testing,microporous filtration of mixing air,microbial monitoring,frequent regeneration to minimize and control microorganism growth,sizing the equipment for suitable water flow,and use of elevated temperatures.Regeneration piping for mixed bed units should be configured to ensure that regeneration chemicals contact all internal surfaces and resins.Rechargeable canisters can be the source of contamination and should be carefully monitored.Full knowledge of previous resin use,minimum storage time between regeneration and use,and appropriate sanitizing procedures are critical factors ensuring proper performance.

Reverse osmosis (RO)units employ a semipermeable membrane and a substantial pressure differential to drive water through the membrane to achieve chemical,microbial,and endotoxin quality improvement.The process streams consist of supply water,product water (permeate),and waste water (reject).Pretreatment and system configuration variations may be necessary,depending on source water to achieve desired performance and reliability.Concerns associated with the design and operation of ROunits include membrane material sensitivity to bacteria and sanitizing agents,membrane fouling,membrane integrity,seal integrity,and the volume of waste water.Failure of membrane or seal integrity will result in product water contamination.Methods of control consist of suitable pretreatment of the water stream,appropriate membrane material selection,integrity challenges,membrane design such as spiral wound to promote flushing action,periodic sanitization,monitoring of differential pressures,conductivity,microbial levels,and total organic carbon.The configuration of the ROunit offers control opportunities by expanding the single-pass scheme to parallel-staged,reject-staged,two-pass,and combination designs.An example would be the use of a two-pass design to improve reliability,quality,and efficiency.ROunits can be used alone or in combination with DIand EDIunits for operational and quality enhancements.

Ultrafiltration is another technology that uses a permeable membrane,but unlike ROit works by mechanical separation rather than osmosis.Because of the filtration ability of the membrane,macromolecular and microbial impurities,such as endotoxins,are reduced.This technology may be appropriate as an intermediate or final purification step.As with RO,successful performance is dependent upon other system unit operations and system configuration.

Issues of concern include compatibility of membrane material with sanitizing agents,membrane integrity,fouling by particles and microorganisms,cartridge contaminant retention,and seal integrity.Control measures include sanitization,designs capable of flushing the membrane surface,integrity challenges,regular cartridge changes,elevated feed water temperature,and monitoring total organic carbon and differential pressure.Additional flexibility in operation is possible through arrangement of the units,such as parallel or series configuration.Care should be taken to avoid stagnant water conditions that could promote microorganism growth in backup or standby units.

Microbial retentive filters (membrane filters)prevent the passage of microorganisms and very small particles.They are used in tank air and inert gas vents and for filtration of compressed air gases used in the regeneration of mixed-bed deionization units.Areas of concern are blockage of tank vents by condensed water vapor,which can cause mechanical damage to the tank,and concentration of microorganisms on the surface of the membrane filter,creating the potential for contamination of the tank or deionizer contents.Control measures include the use of hydrophobic filters and heat tracing vent filter housings to prevent vapor condensation.Sterilization of the unit prior to initial use and periodically thereafter or regular filter changes are also recommended control methods.Microbial retentive filters are sometimes incorporated into purification systems or into water distribution piping.This application should be carefully controlled because,as noted above,these units can become a source for microbial contamination.The potential exists for the release of microorganisms should the membrane filter rupture or as a result of microbial grow-through.Other means of controlling microorganisms and fine particles can be employed in place of membrane filters in the purification and distribution section of water systems.Filters that are intended to be microretentive should be sanitized and integrity tested prior to initial use and at appropriate intervals thereafter.

Positively charged filter media reduce endotoxin levels by electrostatic attraction and adsorption.Application may be related to the unit operation or distribution system,depending upon the microbial control requirements.Filter media that are microbial retentive require the same concerns and controls as indicated in the previous paragraph.Concerns include flow rate,membrane and seal integrity,and retention capacity,which can be affected by the development of a finite charge potential on the filter.Control measures include monitoring differential pressure and endotoxin levels,proper sizing,testing membrane integrity,and configuring units in series to control breakthrough.

Distillation units provide chemical and microbial purification via thermal vaporization,mist elimination,and condensing.Avariety of designs are available,including single-effect,multiple-effect,and vapor compression.The latter two configurations are normally used in larger systems because of their generating capacity and efficiency.Distilled water systems may require less rigorous control of feed water quality than do membrane systems.Areas of concern include carryover of impurities,evaporator flooding,stagnant water,pump and compressor seal design,and conductivity (quality)variations during startup and operation.Methods of control consist of reliable mist elimination,visual or automated high-water-level indication,use of sanitary pumps and compressors,proper drainage,blow down control,and use of on-line conductivity sensing with automated diversion of unacceptable quality water to the waste stream.

Storage tanks are included in water distribution systems to optimize processing equipment capacity.Storage also allows for routine maintenance while maintaining continuous supply to meet manufacturing needs.Design and operation considerations are needed to prevent the development of biofilm,to minimize corrosion,to aid in the use of chemical sanitization of the tanks,and to safeguard mechanical integrity.These considerations may include using closed tanks with smooth interiors and the ability to spray the tank head space.This minimizes corrosion and biofilm development and aids in sanitizing thermally or chemically.

Storage tanks require venting to compensate for the dynamics of changing water levels.This can be accomplished with a hydrophobic microbial retentive membrane filter fitted onto an atmospheric vent.Alternatively,an automatic membrane-filtered compressed gas pressurization and venting system may be used.Rupture disks equipped with a rupture alarm device serve as a further safeguard for the mechanical integrity of the tank.

Distribution configuration should allow for the continuous flow of water in the piping by means of recirculation or should provide for the periodic flushing of the system.Experience has shown that continuously recirculated systems are easier to maintain.

Pumps should be designed to deliver fully turbulent flow conditions to retard the development of biofilms.Components and distribution lines should be sloped and fitted with drain points so that the system can be completely drained.In distribution systems,where the water is circulated at a high temperature,dead legs and low-flow conditions should be avoided,and valved tie-in points should have length-to-diameter ratios of 6or less.In ambient temperature distribution systems,particular care should be exercised to avoid pocket areas and provide for complete drainage.Water exiting from a loop should not be returned to the system.Distribution design should include the placement of sampling valves in the storage tank and at other locations such as the return line of the recirculating water system.The primary sampling site for water should be the valves that deliver water to the point of use.Direct connections to processes or auxiliary equipment should be designed to prevent reverse flow into the controlled water system.The distribution system should permit sanitization for microorganism control.The system may be continuously operated at sanitizing conditions or sanitized periodically.

INSTALLATION AND MATERIALS OF CONSTRUCTION AND COMPONENT SELECTION

Installation techniques are important because they can affect the mechanical,corrosive,and sanitary integrity of the system.Valve installation attitude should promote gravity drainage.Pipe supports should provide appropriate slopes for drainage and should be designed to support the piping adequately under worst-case thermal conditions.Methods of connecting system components,including units of operation,tanks,and distribution piping,require careful attention to preclude potential problems.

Stainless steel welds should provide reliable joints that are internally smooth and corrosion-free.Low-carbon stainless steel,compatible wire filler where necessary,inert gas,automatic welding machines,and regular inspection and documentation help to ensure acceptable weld quality.Follow-up cleaning and passivation are important for removing contamination and corrosion products and to reestablish the passive corrosion-resistant surface.Plastic materials can be fused (welded)in some cases and also require smooth,uniform internal surfaces.Adhesives should be avoided because of the potential 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 fittings.

Materials of construction should be selected to be compatible with control measures such as sanitizing,cleaning,and passivating.Temperature rating is a critical factor in choosing appropriate materials because surfaces may be required to handle elevated operating and sanitization temperatures.Should chemicals or additives be used to clean,control,or sanitize the system,materials resistant to these chemicals or additives must be used.

Materials should be capable of handling turbulent flow and elevated velocities without wear on the corrosive barrier impact,such as the passivation-related chromium oxide surface of stainless steel.The finish on metallic materials such as stainless steel,whether a refined mill finish,polished to a specific grit,or an electropolished treatment,should complement system design and provide satisfactory corrosion and microbial activity resistance.Auxiliary equipment and fittings that require seals,gaskets,diaphragms,filter media,and membranes should exclude materials that permit the possibility of extractables,shedding,and microbial activity.

Insulating materials exposed to stainless steel surfaces should be free of chlorides to avoid the phenomenon of stress corrosion cracking,which can lead to system contamination and the destruction of tanks and critical system components.

Specifications are important to ensure proper selection of materials and to serve as a reference for system qualification and maintenance.Information such as mill reports for stainless steel and reports of composition,ratings,and material handling capabilities for nonmetallic substances should be reviewed for suitability and retained for reference.

Component (auxiliary equipment) selection should be made with assurance that it does not create a source for contamination intrusion.Heat exchangers should be double tube sheet or concentric tube design.They should include differential pressure monitoring or use heat transfer medium of equal or better quality to avoid problems should leaks develop.Pumps should be of sanitary design with seals that prevent contamination of the water.Valves should have smooth internal surfaces with the seat and closing device exposed to the flushing action of water,such as occurs in diaphragm valves.Valves with pocket areas or closing devices (e.g.,ball,plug,gate,globe)that move into and out of a flow area should be avoided.

SANITIZATION

Microbial control in water systems is achieved primarily through sanitization practices.Systems can be sanitized using either thermal or chemical means.In-line UVlight at a wavelength of 254nm can also be used to “sanitize”water in the system continuously.

Thermal approaches to system sanitization include periodic or continuously circulating 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.Although thermal methods control biofilm development,they are not effective in removing established biofilms.

Chemical methods,where compatible,can be used on a wider variety of construction materials.These methods typically employ oxidizing agents such as halogenated compounds,hydrogen peroxide,ozone,or peracetic acid.Halogenated compounds are effective sanitizers but are difficult to flush from the system and tend to leave biofilms intact.Compounds such as hydrogen peroxide,ozone,and peracetic acid oxidize bacteria and biofilms by forming reactive peroxides and free radicals (notably hydroxyl radicals).The short half-life of these compounds,particularly ozone,may require that it be added continuously during the sanitization process.Hydrogen peroxide and ozone rapidly degrade to water and oxygen;peracetic acid degrades to acetic acid in the presence of UVlight.

UVlight impacts on the development of biofilms by reducing the rate of new microbial colonization in the system;however,it is only partially effective against planktonic microorganisms.Alone,UVlight is not an effective tool because it does not eliminate existing biofilm.However,when coupled with conventional thermal or chemical sanitization technologies,it is most effective and can prolong the interval between system sanitizations.The use of UVlight also facilitates the degradation of hydrogen peroxide and ozone.

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 methods 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 established should be such that the system operates in a state of microbiological control and does not exceed Alert Levels.

OPERATION,MAINTENANCE,AND CONTROL

Apreventive maintenance program should be established to ensure that the water system remains in a state of control.The program should include (1)procedures for operating the system,(2)monitoring programs for critical quality attributes and operating conditions,including calibration of critical instruments,(3)a schedule for periodic sanitization,(4)preventive maintenance of components,and (5)control of changes to the mechanical system and to operating conditions.

Operating Procedures— Procedures for operating the water system and performing routine maintenance and corrective action should be written,and they should also define the point when action is required.The procedures should be well documented,detail the function of each job,assign who is responsible for performing the work,and describe how the job is to be conducted.

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

Sanitization— Depending on system design and the selected units of operation,routine periodic sanitization may be necessary to maintain the system in a state of microbial control.Technologies for sanitization are described above.

Preventive Maintenance— Apreventive maintenance program should be in effect.The program should establish what preventive maintenance is to be performed,the frequency of maintenance work,and how the work should be documented.

Change Control— The mechanical configuration and operating conditions must be controlled.Proposed changes should be evaluated for their effects on the whole system.The need to requalify the system after changes are made should be determined.Following a decision to modify a water system,the affected drawings,manuals,and procedures should be revised.

SAMPLING CONSIDERATIONS

Water systems should be monitored frequently enough to ensure that the system is in control and continues to produce water of acceptable quality.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.For example,systems for Water for Injection,because of their more critical microbiological requirements,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 containing chemical sanitizing agents require neutralization prior to microbiological analysis.Samples for microbiological analysis should be tested immediately or suitably protected to preserve the sample until analysis can begin.

Samples of flowing water are only indicative of the concentration of planktonic (free-floating)microorganisms present in the system.Benthic (attached)microorganisms present as biofilms are generally present in greater numbers and are the source of the planktonic population.Microorganisms in biofilms represent a continuous source of contamination and are difficult to sample and quantify.Consequently,the planktonic population is used as an indicator of system contamination levels and is the basis for system Alert Levels.The consistent appearance of elevated planktonic levels is usually an indication of advanced biofilm development in need of remedial control.System control and sanitization are key in controlling biofilm formation and consequent planktonic population.

MICROBIAL CONSIDERATIONS

The major exogenous source of microbial contamination is source or feed water.Feed water quality must,at a minimum,meet the quality attributes of drinking water for which the level of coliforms are regulated.Awide variety of other microorganisms,chiefly Gram-negative bacteria,may be present.These microorganisms 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 to minimize microbial contamination from these sources.

Unit operations can be a major source of endogenous microbial contamination.Microorganisms present in feed water may adsorb to carbon beds,deionizer resins,filter membranes,and other unit operation surfaces and initiate the formation of a biofilm.Biofilm is an adaptive response by certain microorganisms to survive in a low-nutrient environment.Microorganisms in a biofilm are protected from the action of many biocides.Downstream colonization can occur when microorganisms are sloughed off and carried in other areas of the water system.Microorganisms may also attach to suspended particles such as carbon-bed fines and serve as a source of contamination to subsequent purification equipment and distribution systems.

Another source of endogenous microbial contamination is the distribution system.Microorganisms can colonize pipe surfaces,valves,and other areas.There they proliferate,forming a biofilm,which then provides a continuous source of microbial contamination.

Endotoxins are lipopolysaccharides from the cell envelope that is external to the cell wall of Gram-negative bacteria.Gram-negative bacteria readily form biofilms that can become a source of endotoxins.Endotoxins may be associated with either living microorganisms or fragments of dead microorganisms,or they may be free molecules.The free form of endotoxins may be released from cell surfaces or biofilms that colonize the water system,or they may enter the water system via the feed water.Endotoxin levels may be minimized by controlling the introduction of microorganisms and microbial proliferation in the system.This may be accomplished through the normal exclusion or removal action afforded by various unit operations within the treatment system as well as through system sanitization.Other control methods include the use of ultrafilters or charge-modified filters,either in-line or at the point of use.The presence of endotoxins may be monitored as described in the chapter Bacterial Endotoxins Test á85ñ.

Methodological Considerations*

The objective of a water system microbiological monitoring program is to provide sufficient information to control the microbiological quality of the water produced.Product quality requirements should dictate water quality needs.An appropriate level of control may be maintained by using data trending techniques and limiting specific contraindicated microorganisms.Consequently,it may not be necessary to detect all the microorganisms present.The monitoring program and methodology should indicate adverse trends and detect microorganisms that are potentially harmful to the finished product or consumer.

Final selection of method variables should be based on the individual requirements of the system being monitored.It should be recognized that there is no single method capable of detecting all the potential microbial contaminants of a water system.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.

Several criteria should be considered when selecting a method to monitor the microbial content of a pharmaceutical water system.These include method sensitivity,range of organisms recovered,sample throughput,incubation period,cost,and technical complexity.An additional consideration is the use of the classical culture approaches vs.a sophisticated instrument approach.

THE CLASSICAL CULTURE APPROACH

Classical culture approaches for microbial testing of water include but are not limited to pour plates,spread plates,membrane filtration,and most-probable-number (MPN)tests.These methods are generally easy to perform,are less expensive,and provide excellent sample processing throughput.Method sensitivity can be increased via the use of larger sample sizes.This strategy is used in the membrane filtration method.

Culture approaches are further defined by the type of medium used in combination with the incubation temperature and duration.This combination should be selected according to the monitoring needs presented by a specific water system as well as its ability to recover microorganisms that could have a detrimental effect on the product or process.

Two basic forms of media are available for traditional microbiological analysis:“high”nutrient and “low”nutrient.High-nutrient media are intended as general media for the isolation and enumeration of heterotrophic bacteria.Low-nutrient media are beneficial for isolating slow-growing bacteria and bacteria that have been injured by previous exposure to disinfectants and sanitizers such as chlorine.Low-nutrient media may be compared to high-nutrient media,especially during the validation of a water system,to determine whether any additional numbers or types of bacteria are present so that their effect on the end use may be assessed.Additionally,the efficacy of system controls and sanitization on these slower-growing or impaired bacteria can also be assessed.

Duration and temperature of incubation are also critical aspects of a microbiological test method.Classical methodologies using high-nutrient media have required incubation at 30

Cto 35

Cfor 48to 72hours.In certain water systems,incubation at lower temperatures (e.g.,20

Cto 25

C)and longer periods (e.g.,5to 7days)can produce higher counts when compared to classical methods.Whether a particular system needs to be monitored using lower incubation temperatures or longer incubation times should be determined during system validation.

The decision to use longer incubation periods should be made after considering the need for timely information and the type of corrective actions required when an Alert or Action Level is exceeded.The advantages gained by incubating for longer times—recovery of injured microorganisms—slow growers,or more fastidious microorganisms,should be balanced against the need to have a timely investigation and to take corrective action,as well as the ability of these microorganisms to detrimentally affect products or processes.

INSTRUMENT APPROACH

Examples of instrument approaches include microscopic direct counting techniques (e.g.,epifluorescence and immunofluorescence)and radiometric,impedometric,and biochemically based methodologies.These methods all possess a variety of advantages and disadvantages.

One advantage is their precision and accuracy.In general,instrument approaches often have a shorter lead time for obtaining results,which facilitates timely system control.This advantage,however,is often counterbalanced by limited sample processing throughput due to labor-intensive sample processing or other instrument limitations.In addition,instrumental approaches are destructive in that further isolate manipulation for characterization purposes is precluded.Generally,some form of microbial isolate characterization may be a required element of water system monitoring.Consequently,culturing approaches have traditionally been preferred over instrumental approaches because they offer a balance of desirable test attributes and posttest capabilities.

RECOMMENDED METHODOLOGIES

The following general methods obtained from Standard Methods for the Examination of Water and Wastewater,18th Edition,American Public Health Association,Washington,DC20005,are considered appropriate for establishing trends in the number of colony-forming units observed in the routine microbiological monitoring of ingredient water.It is recognized,however,that other combinations of media,time,and temperature of incubation may occasionally or even consistently result in higher numbers of colony-forming units being observed.The extended incubation periods that are usually required by some of the alternative methods available offer disadvantages that may outweigh the higher counts obtained.The somewhat higher baseline counts would not necessarily have greater utility in detecting an excursion or a trend.

Methodologies that can be recommended as generally satisfactory for monitoring pharmaceutical water systems are as follows.

Drinking Water:	POUR PLATE METHOD Minimum sample—1.0mL Plate count agar 42to 72hours incubation at 30C to 35C
Purified Water:	POUR PLATE METHOD Minimum sample—1.0mL Plate count agar 48to 72hours incubation at 30C to 35C
Water for Injection:	MEMBRANE FILTRATION METHOD Minimum sample—100mL Plate count agar 48to 72hours incubation at 30Cto 35C

Identification of Microorganisms

Identifying the isolates recovered from water-monitoring methods may be important in instances where specific waterborne microorganisms may be detrimental to the products or processes in which the water is used.Microorganism information such as this may also be useful when identifying the source of microbial contamination in a product or process.

Often a limited group of microorganisms are continuously recovered from a water system.After repeated characterization,an experienced microbiologist may become proficient at their identification from only a few traits such as colonial morphology and staining characteristics.This level of characterization is adequate for most situations.

Alert and Action Levels

The individual monographs Purified Waterand Water for Injectiondo not include specific microbial limits.These were intentionally omitted,since most current microbiological techniques available require at least 48hours to obtain definitive results.By that time,the water from which the sample was taken has already been employed in the production process.Failure to meet a compendial specification would require rejecting the product lot involved,and this is not the intent of an Alert or Action guideline.The establishment of quantitative microbiological guidelines for water for pharmaceutical purposes is in order because such guidelines will establish procedures to be implemented if that significant excursions beyond these limits occur.

Water systems should be microbiologically monitored to confirm that they continue to operate within their design specifications and produce water of acceptable quality.Monitoring data may be compared to established process parameters or product specifications.Arefinement in the use of process parameters and product specifications is the establishment of Alert and Action Levels,which signal a shift in process performance.Alert and Action Levels are distinct from process parameters and product specifications in that they are used for monitoring and control rather than accept or reject decisions.

Alert Levelsare levels or ranges that,when exceeded,indicate that a process may have drifted from its normal operating condition.Alert Levels constitute a warning and do not necessarily require a corrective action.

Action Levelsare levels or ranges that,when exceeded,indicate that a process has drifted from its normal operating range.Exceeding an Action Level indicates that corrective action should be taken to bring the process back into its normal operating range.

Alert and Action Levels are established within process and product specification tolerances and are based on a combination of technical and product-related considerations.Consequently,exceeding an Alert or Action Level does not imply that product quality has been compromised.

Technical considerations used to establish Alert and Action Levels should include a review of equipment design specifications to ensure that the purification equipment is capable of achieving the required level of purity.In addition,samples should be collected and analyzed over a period of time to develop data reflecting normal water quality trends.Historical or statistically based levels can be established using these data.Levels established in this way measure process performance and are independent of product concerns.

Product-related Alert and Action Levels should represent both product-quality concerns and the ability to effectively manage the purification process.These levels are typically based on a review of process data and an assessment of product sensitivity to chemical and microbiological contamination.The assessment of product susceptibility might include preservative efficacy,water activity,pH,etc.The levels set should be such that,when it is exceeded,product quality is not compromised.

Monitoring data should be analyzed on an ongoing basis to ensure that the process continues to perform within acceptable limits.An analysis of data trends is often used to evaluate process performance.This information can be used to predict departures from established operating parameters,thereby signaling the need for appropriate preventive maintenance.

It should be recognized that the microbial Alert and Action Levels established for any pharmaceutical water system are necessarily linked to the monitoring method chosen.Using the recommended methodologies,generally considered appropriate Action Levels are 500cfu per mLfor Drinking Water,100cfu per mLfor Purified Waterand 10cfu per 100mLfor Water for Injection.

It should be emphasized that the above action guidelines are not intended to be totally inclusive for every situation in which ingredient waters are employed.For example,Gram-negative microorganisms are not excluded from ingredient waters,nor is the presence of Gram-negative microorganisms prohibited in Drinking Waterin the Federal Regulations.The reason for this is that these microorganisms are ubiquitous to the aqueous environment and their exclusion would likely require a sterilization process that would not be appropriate or feasible in many manufacturing scenarios.However,there are situations in which they might not be tolerated:in topical products and in some oral dosage forms.It is,therefore,incumbent upon the manufacturer to supplement the general action guidelines to fit each particular manufacturing situation.

* For additional guidance concerning microbial-water testing methodology,consult Standard Methods for the Examination of Water and Wastewater,18th Edition,American Public Health Association,Washington DC20005.