1231 Water for Pharmaceutical Purposes Usp35

886 ⟨1230⟩ Water for Hemodialysis Applications / General Information USP 35

chemicals used in water treatment. These components Health Association’s Standard Methods for the Examination ofshould be monitored in Water for Hemodialysis being Water and Wastewater, 21st Edition,1 those referenced in theproduced in accordance with established standard op- U.S. Environmental Protection Agency’s Methods for the De-erating procedures. The maximum acceptable levels termination of Metals in Environmental Samples,2 or equiva-of these and other elements and compounds, as pro- lent methods as referenced in ANSI/AAMI RD 62:2006.posed by AAMI (Association for the Advancement ofMedical Instrumentation) are listed in Table 1. These

MICROBIAL CONSIDERATIONSattributes should be periodically monitored to ensurethey are being controlled by the routine testing per-

The Water for Hemodialysis monograph includes total aero-formed in accordance with the Water for Hemodialysisbic microbial count (TAMC) limits of 100 cfu/mL and endo-monograph.toxin limits of 1 USP Endotoxin Unit/mL. In addition, the(2) A comprehensive validation testing of the system pro-absence of Pseudomonas aeruginosa should be routinely de-ducing Water for Hemodialysis should be performedtermined because this is an opportunistic pathogen hazard-initially and periodically thereafter to ensure that theous to acutely ill hemodialysis patients. Both the high mi-water treatment equipment and system sanitizationcrobial counts and the presence of Pseudomonas aeruginosaprocesses are functioning properly.can be associated with inadequate water system mainte-nance and sanitization. Sampling the water should be done

Table 1. Maximum Allowable Chemical Levels in Water for at all use points where the water enters the dialysis equip-Hemodialysis ment. Samples should be assayed within 30 minutes of col-

(water used to prepare dialysate and concentrates from powder at a lection or immediately refrigerated and then assayed withindialysis facility and to reprocess dialyzers for multiple use)* 24 hours of collection. The microbial enumeration and ab-

Maximum Concentration sence tests are performed using procedures found in theElement or Compound (mg/L) USP general test chapters Microbial Enumeration Tests ⟨61⟩

and Tests for Specified Microorganisms ⟨62⟩. Quantification ofContaminants with documented toxicitybacterial endotoxins is performed using procedures found in in hemodialysisthe USP general test chapter Bacterial Endotoxins Test ⟨85⟩.Aluminum 0.01

Because of the incubation time required to obtain defini-Chloramines 0.1 tive microbiological results, water systems should be micro-Free chlorine 0.5 biologically monitored to confirm that they continue to pro-Copper 0.1 duce water of acceptable quality. “Alert” and “ActionFluoride 0.2 Levels” are therefore necessary for the monitoring and con-

trol of the system. An Alert Level constitutes a warning andLead 0.005does not require a corrective action. An Action Level indi-Nitrate (as N) 2cates a drift from normal operating conditions and requires

Sulfate 100 that corrective action be taken to bring the process backZinc 0.1 into the normal operating range. Exceeding an Alert or Ac-

Contaminants normally included in dialysate tion Level does not imply that water quality has been com-Calcium 2 (0.1 mEq/L) promised. The maximum recommended Action Level for a

total viable microbial count in the product water should beMagnesium 4 (0.3 mEq/L)no greater than 25 cfu/mL, and the maximum recom-Potassium 8 (0.2 mEq/L)mended Action Level for bacterial endotoxins should be no

Sodium 70 (3.0 mEq/L) greater than 0.25 USP Endotoxin Unit/mL. As with all pro-Other contaminants cess control values, Action and Alert Levels should be estab-

Antimony 0.006 lished from normal system monitoring trends and processArsenic 0.005 capabilities in a fashion that allows remedial actions to occur

in response to process control level excursions well beforeBarium 0.1specifications are exceeded (also see Microbial ConsiderationsBeryllium 0.0004under Water for Pharmaceutical Purposes ⟨1231⟩).

Cadmium 0.001Chromium 0.014

Mercury 0.0002Selenium 0.09Silver 0.005 ⟨1231⟩ WATER FORThallium 0.002 PHARMACEUTICAL PURPOSES

*Reprinted with permission from ANSI/AAMI RD62: 2006, “Water treat-ment equipment for hemodialysis applications”, Association for theAdvancement of Medical Instrumentation, Arlington, VA.

INTRODUCTIONThe chemical limits included in Table 1 have been recog-nized by federal government agencies as standards for

Water is widely used as a raw material, ingredient, andWater for Hemodialysis. Written standard operating proce-solvent in the processing, formulation, and manufacture ofdures for water testing should be established by the physi-pharmaceutical products, active pharmaceutical ingredientscian in charge or the designated facility manager. The test(APIs) and intermediates, compendial articles, and analyticalfrequency decision should be based upon historical datareagents. This general information chapter provides addi-analysis, the quality of the source water as reported by thetional information about water, its quality attributes that aremunicipal water treatment facility or public health agency innot included within a water monograph, processing tech-the area, etc. Records should be maintained to document

levels and any necessary remedial action taken promptly. 1American Public Health Association, Washington, DC 20005.Chemical analysis of water components listed should be 2U.S. Environmental Protection Agency Publication EPA-600-R-94-111, Cincin-

nati, OH.performed using methods referenced in the American Public

Official from May 1, 2012Copyright (c) 2012 The United States Pharmacopeial Convention. All rights reserved.

Accessed from 24.232.240.237 by cardinalpts on Sun May 27 19:34:17 EDT 2012

USP 35 General Information / ⟨1231⟩ Water for Pharmaceutical Purposes 887

niques that can be used to improve water quality, and a SOURCE OR FEED WATER CONSIDERATIONSdescription of minimum water quality standards that shouldbe considered when selecting a water source. To ensure adherence to certain minimal chemical and mi-

This information chapter is not intended to replace ex- crobiological quality standards, water used in the produc-isting regulations or guides that already exist to cover USA tion of drug substances or as source or feed water for theand International (ICH or WHO) GMP issues, engineering preparation of the various types of purified waters mustguides, or other regulatory (FDA, EPA, or WHO) guidances meet the requirements of the National Primary Drinkingfor water. The contents will help users to better understand Water Regulations (NPDWR) (40 CFR 141) issued by thepharmaceutical water issues and some of the microbiologi- U.S. Environmental Protection Agency (EPA) or the drinkingcal and chemical concerns unique to water. This chapter is water regulations of the European Union or Japan, or thenot an all-inclusive writing on pharmaceutical waters. It con- WHO drinking water guidelines. Limits on the types andtains points that are basic information to be considered, quantities of certain organic and inorganic contaminants en-when appropriate, for the processing, holding, and use of sure that the water will contain only small, safe quantities ofwater. It is the user’s responsibility to assure that pharma- potentially objectionable chemical species. Therefore, waterceutical water and its production meet applicable govern- pretreatment systems will only be challenged to removemental regulations, guidances, and the compendial specifi- small quantities of these potentially difficult-to-removecations for the types of water used in compendial articles. chemicals. Also, control of objectionable chemical contami-

Control of the chemical purity of these waters is impor- nants at the source-water stage eliminates the need to spe-tant and is the main purpose of the monographs in this cifically test for some of them (e.g., trihalomethanes andcompendium. Unlike other official articles, the bulk water heavy metals) after the water has been further purified.monographs (Purified Water and Water for Injection) also Microbiological requirements of drinking water ensure thelimit how the article can be produced because of the belief absence of coliforms, which, if determined to be of fecalthat the nature and robustness of the purification process is origin, may indicate the potential presence of other poten-directly related to the resulting purity. The chemical attrib- tially pathogenic microorganisms and viruses of fecal origin.utes listed in these monographs should be considered as a Meeting these microbiological requirements does not ruleset of minimum specifications. More stringent specifications out the presence of other microorganisms, which could bemay be needed for some applications to ensure suitability considered undesirable if found in a drug substance or for-for particular uses. Basic guidance on the appropriate appli- mulated product.cations of these waters is found in the monographs and is To accomplish microbial control, Municipal Water Authori-further explained in this chapter. ties add disinfectants to drinking water. Chlorine-containing

Control of the microbiological quality of water is impor- and other oxidizing substances have been used for manytant for many of its uses. Most packaged forms of water decades for this purpose and have generally been consid-that have monograph standards are required to be sterile ered to be relatively innocuous to humans. However, thesebecause some of their intended uses require this attribute oxidants can interact with naturally occurring organic mat-for health and safety reasons. USP has determined that a ter to produce disinfection by-products (DBPs), such asmicrobial specification for the bulk monographed waters is trihalomethanes (THMs, including chloroform, bromodichlo-inappropriate and has not been included within the romethane, and dibromochloromethane) and haloaceticmonographs for these waters. These waters can be used in a acids (HAAs, including dichloroacetic acid and trichloroace-variety of applications, some requiring extreme microbiolog- tic acid). The levels of DBPs produced vary with the levelical control and others requiring none. The needed micro- and type of disinfectant used and the levels and types ofbial specification for a given bulk water depends upon its organic materials found in the water, which can varyuse. A single specification for this difficult-to-control attri- seasonally.bute would unnecessarily burden some water users with ir- Because high levels of DBPs are considered a health haz-relevant specifications and testing. However, some applica- ard in drinking water, Drinking Water Regulations mandatetions may require even more careful microbial control to their control to generally accepted nonhazardous levels.avoid the proliferation of microorganisms ubiquitous to However, depending on the unit operations used for furtherwater during the purification, storage, and distribution of water purification, a small fraction of the DBPs in the start-this substance. A microbial specification would also be inap- ing water may carry over to the finished water. Therefore,propriate when related to the “utility” or continuous supply the importance of having minimal levels of DBPs in thenature of this raw material. Microbial specifications are typi- starting water, while achieving effective disinfection, iscally assessed by test methods that take at least 48 to 72 important.hours to generate results. Because pharmaceutical waters DBP levels in drinking water can be minimized by usingare generally produced by continuous processes and used in disinfectants such as ozone, chloramines, or chlorine diox-products and manufacturing processes soon after genera- ide. Like chlorine, their oxidative properties are sufficient totion, the water is likely to have been used well before defini- damage some pretreatment unit operations and must betive test results are available. Failure to meet a compendial removed early in the pretreatment process. The completespecification would require investigating the impact and removal of some of these disinfectants can be problematic.making a pass/fail decision on all product lots between the For example, chloramines may degrade during the disinfec-previous sampling’s acceptable test result and a subsequent tion process or during pretreatment removal, thereby releas-sampling’s acceptable test result. The technical and logistical ing ammonia, which in turn can carry over to the finishedproblems created by a delay in the result of such an analysis water. Pretreatment unit operations must be designed anddo not eliminate the user’s need for microbial specifications. operated to adequately remove the disinfectant, drinkingTherefore, such water systems need to be operated and water DBPs, and objectionable disinfectant degradants. A se-maintained in a controlled manner that requires that the rious problem can occur if unit operations designed to re-system be validated to provide assurance of operational sta- move chlorine were, without warning, challenged with chlo-bility and that its microbial attributes be quantitatively ramine-containing drinking water from a municipality thatmonitored against established alert and action levels that had been mandated to cease use of chlorine disinfection towould provide an early indication of system control. The comply with ever tightening EPA Drinking Water THM speci-issues of water system validation and alert/action levels and fications. The dechlorination process might incompletely re-specifications are included in this chapter. move the chloramine, which could irreparably damage

downstream unit operations, but also the release of ammo-nia during this process might carry through pretreatmentand prevent the finished water from passing compendialconductivity specifications. The purification process must be



888 ⟨1231⟩ Water for Pharmaceutical Purposes / General Information USP 35

reassessed if the drinking water disinfectant is changed, em- The Purified Water monograph also allows bulk packagingphasizing the need for a good working relationship between for commercial use elsewhere. There is a potential for thethe pharmaceutical water manufacturer and the drinking occurrence of microbial contamination and other qualitywater provider. changes in this bulk packaged nonsterile water. Therefore,

this form of Purified Water should be prepared and stored ina fashion that limits microbial growth and/or simply used in

TYPES OF WATER a timely fashion before microbial proliferation renders it un-suitable for its intended use. Also depending on the material

There are many different grades of water used for phar- used for packaging, there could be extractable compoundsmaceutical purposes. Several are described in USP leaching into the water from the packaging. Though thismonographs that specify uses, acceptable methods of prep- article may meet its required chemical attributes, such ex-aration, and quality attributes. These waters can be divided tractables may render the water an inappropriate choice forinto two general types: bulk waters, which are typically pro- some applications. It is the user’s responsibility to ensureduced on site where they are used; and sterile waters, which fitness for use of this packaged article when used in manu-are produced, packaged, and sterilized to preserve microbial facturing, clinical, or analytical applications where the purequality throughout their packaged shelf life. There are sev- bulk form of the water is indicated.eral specialized types of sterile waters, differing in their des- Water for Injection—Water for Injection (see the USPignated applications, packaging limitations, and other qual- monograph) is used as an excipient in the production ofity attributes. parenteral and other preparations where product endotoxinThere are also other types of water for which there are no content must be controlled, and in other pharmaceuticalmonographs. These are all bulk waters, with names given applications, such as cleaning of certain equipment and par-for descriptive purposes only. Many of these waters are used enteral product-contact components. The minimum qualityin specific analytical methods. The associated text may not of source or feed water for the generation of Water for Injec-specify or imply certain quality attributes or modes of prep- tion is Drinking Water as defined by the U.S. EPA, EU, Japan,aration. These nonmonographed waters may not necessarily or WHO. This source water may be pretreated to render itadhere strictly to the stated or implied modes of preparation suitable for subsequent distillation (or whatever other vali-or attributes. Waters produced by other means or controlled dated process is used according to the monograph). Theby other test attributes may equally satisfy the intended uses finished water must meet all of the chemical requirementsfor these waters. It is the user’s responsibility to ensure that for Purified Water as well as an additional bacterial endotoxinsuch waters, even if produced and controlled exactly as specification. Since endotoxins are produced by the kinds ofstated, be suitable for their intended use. Wherever the term microorganisms that are prone to inhabit water, the equip-“water” is used within this compendia without other de- ment and procedures used by the system to purify, store,scriptive adjectives or clauses, the intent is that water of no and distribute Water for Injection must be designed to mini-less purity than Purified Water be used. mize or prevent microbial contamination as well as removeWhat follows is a brief description of the various types of incoming endotoxins from the starting water. Water for In-pharmaceutical waters and their significant uses or attrib- jection systems must be validated to reliably and consistentlyutes. Figure 1 may also be helpful in understanding some of produce and distribute this quality of water.the various types of waters. The Water for Injection monograph also allows it to be

packed in bulk for commercial use. Bulk packaged Water forInjection is required to be sterile, thus eliminating microbialBulk Monographed Waters and Steamcontamination quality changes. However, packaging extract-ables may render this water an inappropriate choice forThe following waters are typically produced in large vol-some applications. It is the user’s responsibility to ensureume by a multiple-unit operation water system and distrib-fitness for use of this packaged article when used in manu-uted by a piping system for use at the same site. Thesefacturing, clinical, or analytical applications where the purerparticular pharmaceutical waters must meet the quality at-bulk form of the water is indicated.tributes as specified in the related monographs.

Water for Hemodialysis—Water for Hemodialysis (see thePurified Water—Purified Water (see the USP monograph)USP monograph) is used for hemodialysis applications, pri-is used as an excipient in the production of nonparenteralmarily the dilution of hemodialysis concentrate solutions. Itpreparations and in other pharmaceutical applications, suchis produced and used on site and is made from EPA Drink-as cleaning of certain equipment and nonparenteral prod-ing Water which has been further purified to reduce chemi-uct-contact components. Unless otherwise specified, Purifiedcal and microbiological components. It may be packagedWater is also to be used for all tests and assays for whichand stored in unreactive containers that preclude bacterialwater is indicated (see General Notices and Requirements).entry. The term “unreactive containers” implies that thePurified Water is also referenced throughout the USP–NF. Re-container, especially its water contact surfaces, are notgardless of the font and letter case used in its spelling,changed in any way by the water, such as by leaching ofwater complying with the Purified Water monograph is in-container-related compounds into the water or by anytended. Purified Water must meet the requirements for ionicchemical reaction or corrosion caused by the water. Theand organic chemical purity and must be protected fromwater contains no added antimicrobials and is not intendedmicrobial contamination. The minimal quality of source orfor injection. Its attributes include specifications for Waterfeed water for the production of Purified Water is Drinkingconductivity, Total organic carbon (or oxidizable substances),Water. This source water may be purified using unit opera-Microbial limits, and Bacterial endotoxins. The water conduc-tions that include deionization, distillation, ion exchange, re-tivity and total organic carbon attributes are identical toverse osmosis, filtration, or other suitable purification proce-those established for Purified Water and Water for Injection;dures. Purified water systems must be validated to reliablyhowever, instead of total organic carbon, the organic con-and consistently produce and distribute water of acceptabletent may alternatively be measured by the test for Oxidiza-chemical and microbiological quality. Purified water systemsble substances. The Microbial limits attribute for this water isthat function under ambient conditions are particularly sus-unique among the “bulk” water monographs, but is justifiedceptible to the establishment of tenacious biofilms of micro-on the basis of this water’s specific application that has mi-organisms, which can be the source of undesirable levels ofcrobial content requirements related to its safe use. The Bac-viable microorganisms or endotoxins in the effluent water.terial endotoxins attribute is likewise established at a levelThese systems require frequent sanitization and microbiolog-related to its safe use.ical monitoring to ensure water of appropriate microbiologi-

Pure Steam—Pure Steam (see the USP monograph) iscal quality at the points of use.also sometimes referred to as “clean steam”. It is used




Figure 1. Water for pharmaceutical purposes.

where the steam or its condensate would directly contact could arise from entrained source water droplets, anticorro-official articles or article-contact surfaces, such as during sion steam additives, or residues from the steam productiontheir preparation, sterilization, or cleaning where no subse- and distribution system itself. The attributes in the Purequent processing step is used to remove any codeposited Steam monograph should detect most of the contaminantsimpurity residues. These Pure Steam applications include but that could arise from these sources. If the official article ex-are not limited to porous load sterilization processes, prod- posed to potential Pure Steam residues is intended for par-uct or cleaning solutions heated by direct steam injection, enteral use or other applications where the pyrogenic con-or humidification of processes where steam injection is used tent must be controlled, the Pure Steam must additionallyto control the humidity inside processing vessels where the meet the specification for Bacterial Endotoxins ⟨85⟩.official articles or their in-process forms are exposed. The These purity attributes are measured on the condensate ofprimary intent of using this quality of steam is to ensure the article, rather than the article itself. This, of course, im-that official articles or article-contact surfaces exposed to it parts great importance to the cleanliness of the Pure Steamare not contaminated by residues within the steam. condensate generation and collection process because it

Pure Steam is prepared from suitably pretreated source must not adversely impact the quality of the resulting con-water analogously to either the pretreatment used for Puri- densed fluid.fied Water or Water for Injection. The water is vaporized with Other steam attributes not detailed in the monograph, insuitable mist elimination, and distributed under pressure. particular, the presence of even small quantities of noncon-The sources of undesirable contaminants within Pure Steam densable gases or the existence of a superheated or dry




state, may also be important for applications such as sterili- and sterilized in single-dose containers of larger than 1 L inzation. The large release of energy (latent heat of condensa- size that allows rapid delivery of its contents. It need nottion) as water changes from the gaseous to the liquid state meet the requirement under small-volume injections in theis the key to steam’s sterilization efficacy and its efficiency, general test chapter Particulate Matter in Injections ⟨788⟩. Itin general, as a heat transfer agent. If this phase change may also be used in other applications which do not have(condensation) is not allowed to happen because the steam particulate matter specifications, where bulk Water for Injec-is extremely hot and in a persistent superheated, dry state, tion or Purified Water is indicated but where access to a vali-then its usefulness could be seriously compromised. Non- dated water system is not practical, or where somewhatcondensable gases in steam tend to stratify or collect in cer- larger quantities than are provided as Sterile Water for Injec-tain areas of a steam sterilization chamber or its load. These tion are needed.surfaces would thereby be at least partially insulated from Sterile Water for Inhalation—Sterile Water for Inhalationthe steam condensation phenomenon, preventing them (see the USP monograph) is Water for Injection that is pack-from experiencing the full energy of the sterilizing condi- aged and rendered sterile and is intended for use in inhala-tions. Therefore, control of these kinds of steam attributes, tors and in the preparation of inhalation solutions. It carriesin addition to its chemical purity, may also be important for a less stringent specification for bacterial endotoxins thancertain Pure Steam applications. However, because these ad- Sterile Water for Injection and therefore is not suitable forditional attributes are use-specific, they are not mentioned parenteral applications.in the Pure Steam monograph.

Note that less pure “plant steam” may be used for steamsterilization of nonproduct contact nonporous loads, for Nonmonographed Manufacturing Watersgeneral cleaning of nonproduct contact equipment, as anonproduct contact heat exchange medium, and in all com- In addition to the bulk monographed waters describedpatible applications involved in bulk pharmaceutical chemi- above, nonmonographed waters can also be used in phar-cal and API manufacture. maceutical processing steps such as cleaning, synthetic

steps, or a starting material for further purification. The fol-lowing is a description of several of these nonmonographed

Sterile Monographed Waters waters as cited in various locations within this compendia.Drinking Water—This type of water can be referred toThe following monographed waters are packaged forms as Potable Water (meaning drinkable or fit to drink), Na-of either Purified Water or Water for Injection that have been tional Primary Drinking Water, Primary Drinking Water, orsterilized to preserve their microbiological properties. These National Drinking Water. Except where a singular drinkingwaters may have specific intended uses as indicated by their water specification is stated (such as the NPDWR [U.S. Envi-names and may also have restrictions on packaging configu- ronmental Protection Agency’s National Primary Drinkingrations related to those uses. In general, these waters may Water Regulations as cited in 40 CFR Part 141]), this waterbe used in lieu of the bulk form of water from which they must comply with the quality attributes of either thewere derived. However, the user should take into considera- NPDWR, or the drinking water regulations of the Europeantion that the packaging and sterilization processes used for Union or Japan, or the WHO Drinking Water Guidelines. Itthe articles may leach materials from the packaging material may be derived from a variety of sources including a publicinto the water over its shelf life, rendering it less pure than water utility, a private water supply (e.g., a well), or a com-the original water placed into the package. It is the user’s bination of these sources. Drinking Water may be used inresponsibility to ensure fitness for use of this article when the early stages of cleaning pharmaceutical manufacturingused in manufacturing, clinical, or analytical applications equipment and product-contact components. Drinkingwhere the purer bulk form of the water is indicated. Water is also the minimum quality of water that should be

Sterile Purified Water—Sterile Purified Water (see the USP used for the preparation of official substances and othermonograph) is Purified Water, packaged and rendered ster- bulk pharmaceutical ingredients. Where compatible with theile. It is used in the preparation of nonparenteral com- processes, the allowed contaminant levels in Drinking Waterpendial dosage forms or in analytical applications requiring are generally considered safe for use for official substancesPurified Water where access to a validated Purified Water sys- and other drug substances. Where required by the process-tem is not practical, where only a relatively small quantity is ing of the materials to achieve their required final purity,needed, where Sterile Purified Water is required, or where higher qualities of water may be needed for these manufac-bulk packaged Purified Water is not suitably microbiologically turing steps, perhaps even as pure as Water for Injection orcontrolled. Purified Water. Such higher purity waters, however, might

Sterile Water for Injection—Sterile Water for Injection require only selected attributes to be of higher purity than(see the USP monograph) is Water for Injection packaged Drinking Water (see Figure 2). Drinking Water is the pre-and rendered sterile. It is used for extemporaneous prescrip- scribed source or feed water for the production of bulktion compounding and as a sterile diluent for parenteral monographed pharmaceutical waters. The use of Drinkingproducts. It may also be used for other applications where Water specifications establishes a reasonable set of maxi-bulk Water for Injection or Purified Water is indicated but mum allowable levels of chemical and microbiological con-where access to a validated water system is either not prac- taminants with which a water purification system will betical or where only a relatively small quantity is needed. challenged. As seasonal variations in the quality attributes ofSterile Water for Injection is packaged in single-dose contain- the Drinking Water supply can occur, due consideration toers not larger than 1 L in size. its synthetic and cleaning uses must be given. The process-

ing steps in the production of pharmaceutical waters mustBacteriostatic Water for Injection—Bacteriostatic Waterbe designed to accommodate this variability.for Injection (see the USP monograph) is sterile Water for

Injection to which has been added one or more suitable an- Hot Purified Water—This water is used in the prepara-timicrobial preservatives. It is intended to be used as a dilution instructions for USP–NF articles and is clearly intendedent in the preparation of parenteral products, most typically to be Purified Water that has been heated to an unspecifiedfor multi-dose products that require repeated content with- temperature in order to enhance solubilization of other in-drawals. It may be packaged in single-dose or multiple-dose gredients. There is no upper temperature limit for the watercontainers not larger than 30 mL. (other than being less than 100°), but for each monograph

there is an implied lower limit below which the desiredSterile Water for Irrigation—Sterile Water for Irrigationsolubilization effect would not occur.(see the USP monograph) is Water for Injection packaged




Figure 2. Selection of water for pharmaceutical purposes.

tests. Regardless of the original reason for the creation ofNonmonographed Analytical Watersthese numerous special analytical waters, it is possible thatthe attributes of these special waters could now be met byBoth General Notices and Requirements and the introduc-the basic preparation steps and current specifications of Pu-tory section to Reagents, Indicators, and Solutions clearlyrified Water. In some cases, however, some of the cited post-state that where the term “water,” without qualification orprocessing steps are still necessary to reliably achieve theother specification, is indicated for use in analyses, the qual-required attributes.ity of water shall be Purified Water. However, numerous such

Users are not obligated to employ specific and perhapsqualifications do exist. Some of these qualifications involvearchaically generated forms of analytical water where alter-methods of preparation, ranging from specifying the pri-natives with equal or better quality, availability, or analyticalmary purification step to specifying additional purification.performance may exist. The consistency and reliability forOther qualifications call for specific attributes to be met thatproducing these alternative analytical waters should be veri-might otherwise interfere with analytical processes. In mostfied as producing the desired attributes. In addition, anyof these latter cases, the required attribute is not specificallyalternative analytical water must be evaluated on an applica-tested. Rather, a further “purification process” is specifiedtion-by-application basis by the user to ensure its suitability.that ostensibly allows the water to adequately meet this re-Following is a summary of the various types ofquired attribute.nonmonographed analytical waters that are cited in theHowever, preparation instructions for many reagents wereUSP–NF.carried forward from the innovator’s laboratories to the orig-

inally introduced monograph for a particular USP–NF article Distilled Water—This water is produced by vaporizingor general test chapter. The quality of the reagent water liquid water and condensing it in a purer state. It is useddescribed in these tests may reflect the water quality desig- primarily as a solvent for reagent preparation, but it is alsonation of the innovator’s laboratory. These specific water specified in the execution of other aspects of tests, such asdesignations may have originated without the innovator’s for rinsing an analyte, transferring a test material as a slurry,awareness of the requirement for Purified Water in USP–NF as a calibration standard or analytical blank, and for test




apparatus cleaning. It is also cited as the starting water to used as a chromatography reagent, monograph-specified fil-be used for making High Purity Water. Because none of the ter ratings range from 0.5 µm to unspecified.cited uses of this water imply a need for a particular purity High Purity Water—The preparation of this water is de-attribute that can only be derived by distillation, water fined in Containers—Glass ⟨660⟩. It is water that is preparedmeeting the requirements for Purified Water derived by other by deionizing previously distilled water, and then filtering itmeans of purification could be equally suitable where Dis- through a 0.45-µm rated membrane. This water must havetilled Water is specified. an in-line conductivity of not greater than 0.15 µS/cm (6.67

Freshly Distilled Water—Also called “recently distilled Megohm-cm) at 25°. For the sake of purity comparison, thewater”, it is produced in a similar fashion to Distilled Water analogous Stage 1 and 2 conductivity requirements for Puri-and should be used shortly after its generation. This implies fied Water at the same temperature are 1.3 µS/cm and 2.1the need to avoid endotoxin contamination as well as any µS/cm, respectively. The preparation specified in Contain-other adventitious forms of contamination from the air or ers—Glass ⟨660⟩ uses materials that are highly efficientcontainers that could arise with prolonged storage. It is used deionizers and that do not contribute copper ions or organ-for preparing solutions for subcutaneous test animal injec- ics to the water, assuring a very high quality water. If thetions as well as for a reagent solvent in tests for which there water of this purity contacts the atmosphere even briefly asappears to be no particularly high water purity needed that it is being used or drawn from its purification system, itscould be ascribable to being “freshly distilled”. In the “test- conductivity will immediately degrade, by as much as aboutanimal” use, the term “freshly distilled” and its testing use 1.0 µS/cm, as atmospheric carbon dioxide dissolves in theimply a chemical, endotoxin, and microbiological purity that water and equilibrates to bicarbonate ions. Therefore, if thecould be equally satisfied by Water for Injection (though no analytical use requires that water purity remains as high asreference is made to these chemical, endotoxin, or microbial possible, its use should be protected from atmospheric ex-attributes or specific protection from recontamination). For posure. This water is used as a reagent, as a solvent fornonanimal uses, water meeting the requirements for Purified reagent preparation, and for test apparatus cleaning whereWater derived by other means of purification and/or storage less pure waters would not perform acceptably. However, ifperiods could be equally suitable where “recently distilled a user’s routinely available purified water is filtered andwater” or Freshly Distilled Water is specified. meets or exceeds the conductivity specifications of High Pu-

rity Water, it could be used in lieu of High Purity Water.Deionized Water—This water is produced by an ion-exchange process in which the contaminating ions are re- Ammonia-Free Water—Functionally, this water mustplaced with either H+ or OH– ions. Similarly to Distilled have a negligible ammonia concentration to avoid interfer-Water, Deionized Water is used primarily as a solvent for rea- ence in tests sensitive to ammonia. It has been equated withgent preparation, but it is also specified in the execution of High Purity Water that has a significantly tighter Stage 1other aspects of tests, such as for transferring an analyte conductivity specification than Purified Water because of thewithin a test procedure, as a calibration standard or analyti- latter’s allowance for a minimal level of ammonium amongcal blank, and for test apparatus cleaning. Also, none of the other ions. However, if the user’s Purified Water were filteredcited uses of this water imply any needed purity attribute and met or exceeded the conductivity specifications of Highthat can only be achieved by deionization. Therefore, water Purity Water, it would contain negligible ammonia or othermeeting the requirements for Purified Water that is derived ions and could be used in lieu of High Purity Water.by other means of purification could be equally suitable Carbon Dioxide-Free Water—The introductory portionwhere Deionized Water is specified. of the Reagents, Indicators, and Solutions section defines this

Freshly Deionized Water—This water is prepared in a water as Purified Water that has been vigorously boiled for atsimilar fashion to Deionized Water, though as the name sug- least 5 minutes, then cooled and protected from absorptiongests, it is to be used shortly after its production. This im- of atmospheric carbon dioxide. Because the absorption ofplies the need to avoid any adventitious contamination that carbon dioxide tends to drive down the water pH, most ofcould occur upon storage. This water is indicated for use as the uses of Carbon Dioxide-Free Water are either associateda reagent solvent as well as for cleaning. Due to the nature as a solvent in pH-related or pH-sensitive determinations orof the testing, Purified Water could be a reasonable alterna- as a solvent in carbonate-sensitive reagents or determina-tive for these applications. tions. Another use of this water is for certain optical rotation

and color and clarity of solution tests. Though it is possibleDeionized Distilled Water—This water is produced bythat this water is indicated for these tests simply because ofdeionizing (see Deionized Water) Distilled Water. This water isits purity, it is also possible that the pH effects of carbonused as a reagent in a liquid chromatography test that re-dioxide containing water could interfere with the results ofquires a high purity. Because of the importance of this highthese tests. A third plausible reason that this water is indi-purity, water that barely meets the requirements for Purifiedcated is that outgassing air bubbles might interfere withWater may not be acceptable. High Purity Water (see below)these photometric-type tests. The boiled water preparationcould be a reasonable alternative for this water.approach will also greatly reduce the concentrations ofFiltered Distilled or Deionized Water—This water is es- many other dissolved gases along with carbon dioxide.sentially Purified Water produced by distillation or deioniza- Therefore, in some of the applications for Carbon Dioxide-tion that has been filtered through a 1.2-µm rated mem- Free Water, it could be the inadvertent deaeration effect thatbrane. This water is used in particulate matter testing where actually renders this water suitable. In addition to boiling,the presence of particles in the water could bias the test deionization is perhaps an even more efficient process forresults (see Particulate Matter in Injections ⟨788⟩). Because removing dissolved carbon dioxide (by drawing the dis-the chemical water purity needed for this test could also be solved gas equilibrium toward the ionized state with subse-afforded by water purification processes other than distilla- quent removal by the ion-exchange resins). If the startingtion or deionization, filtered water meeting the requirements Purified Water is prepared by an efficient deionization pro-for Purified Water but produced by means other than distilla- cess and protected after deionization from exposure to at-tion or deionization could be equally suitable. mospheric air, water that is carbon dioxide-free can be ef-

Filtered Water—This water is Purified Water that has fectively made without the application of heat. However thisbeen filtered to remove particles that could interfere with deionization process does not deaerate the water, so if Puri-the analysis where the water is used. Where used for prepar- fied Water prepared by deionization is considered as a sub-ing samples for particulate matter testing (see Particulate stitute water in a test requiring Carbon Dioxide-Free Water,Matter in Injections ⟨788⟩), though unspecified in the user must verify that it is not actually water akin tomonographs, water filtration should be through a 1.2-µm Deaerated Water (discussed below) that is needed for thefilter to be consistent with the general test chapter. Where test. As indicated in High Purity Water, even brief contact

with the atmosphere can allow small amounts of carbon




dioxide to dissolve, ionize, and significantly degrade the that is maintained in a hot state and that is inert gas blan-conductivity and lower the pH. If the analytical use requires keted during its preparation and storage and distribution.the water to remain as pH-neutral and as carbon dioxide- Though oxygen is poorly soluble in hot water, such waterfree as possible, even the analysis should be protected from may not be oxygen-free. Whatever procedure used for re-atmospheric exposure. However, in most applications, at- moving oxygen should be verified as reliably producingmospheric exposure during testing does not significantly af- water that is fit for use.fect its suitability in the test. LAL Reagent Water—This water is also referred to as

Ammonia- and Carbon Dioxide-Free Water—As implied endotoxin-free water. This is usually Water for Injection,by the name, this water should be prepared by approaches which may have been sterilized. It is free from a level ofcompatible with those mentioned for both Ammonia-Free endotoxin that would yield any detectable reaction or inter-Water and Carbon Dioxide-Free Water. Because the carbon ference with the Limulus amebocyte lysate reagent used indioxide-free attribute requires post-production protection the Bacterial Endotoxins Test ⟨85⟩.from the atmosphere, it is appropriate to first render the Organic-Free Water—This water is defined by Residualwater ammonia-free using the High Purity Water process fol- Solvents ⟨467⟩ as producing no significantly interfering gaslowed by the boiling and carbon dioxide-protected cooling chromatography peaks. Referenced monographs specify us-process. The High Purity Water deionization process for cre- ing this water as the solvent for the preparation of standardating Ammonia-Free Water will also remove the ions gener- and test solutions for the Residual solvents test.ated from dissolved carbon dioxide and ultimately, by Lead-Free Water—This water is used as a transferringforced equilibration to the ionized state, all the dissolved diluent for an analyte in a Lead ⟨251⟩ test. Though no spe-carbon dioxide. Therefore, depending on its use, an accept- cific instructions are given for its preparation, it must notable procedure for making Ammonia- and Carbon Dioxide- contain any detectable lead. Purified Water should be a suit-Free Water could be to transfer and collect High Purity Water able substitute for this water.in a carbon dioxide intrusion-protected container.

Chloride-Free Water—This water is specified as the sol-Deaerated Water—This water is Purified Water that has vent for use in an assay that contains a reactant that preci-been treated to reduce the content of dissolved air by “suit- pitates in the presence of chloride. Though no specific prep-able means”. In the Reagents section, approaches for boil- aration instructions are given for this water, its rathering, cooling (similar to Carbon Dioxide-Free Water but with- obvious attribute is having a very low chloride level in orderout the atmospheric carbon dioxide protection), and to be unreactive with this chloride sensitive reactant. Purifiedsonication are given as applicable for test uses other than Water could be used for this water but should be tested todissolution and drug release testing. Though Deaerated ensure that it is unreactive.Water is not mentioned by name in Dissolution ⟨711⟩, sug-Hot Water—The uses of this water include solvents forgested methods for deaerating dissolution media (which

achieving or enhancing reagent solubilization, restoring themay be water) include warming to 41°, vacuum filteringoriginal volume of boiled or hot solutions, rinsing insolublethrough a 0.45-µm rated membrane, and vigorously stirringanalytes free of hot water soluble impurities, solvents forthe filtrate while maintaining the vacuum. This chapter spe-reagent recrystallization, apparatus cleaning, and as a solu-cifically indicates that other validated approaches may bebility attribute for various USP–NF articles. In only one mon-used. In other monographs that also do not mention Deaer-ograph is the temperature of “hot” water specified; so in allated Water by name, degassing of water and other reagentsthe other cases, the water temperature is less important, butis accomplished by sparging with helium. Deaerated Water isshould be high enough to achieve the desirable effect. In allused in both dissolution testing as well as liquid chromatog-cases, the chemical quality of the water is implied to be thatraphy applications where outgassing could either interfereof Purified Water.with the analysis itself or cause erroneous results due to in-

accurate volumetric withdrawals. Applications where ambi-ent temperature water is used for reagent preparation, but VALIDATION AND QUALIFICATION OFthe tests are performed at elevated temperatures, are candi- WATER PURIFICATION, STORAGE, ANDdates for outgassing effects. If outgassing could interfere

DISTRIBUTION SYSTEMSwith test performance, including chromatographic flow, col-orimetric or photometric measurements, or volumetric accu-

Establishing the dependability of pharmaceutical waterracy, then Deaerated Water should probably be used,purification, storage, and distribution systems requires anwhether called for in the analysis or not. The above deaera-appropriate period of monitoring and observation. Ordinar-tion approaches might not render the water “gas-free”. Atily, few problems are encountered in maintaining the chem-best, they reduce the dissolved gas concentrations so thatical purity of Purified Water and Water for Injection. Neverthe-outgassing caused by temperature changes is not likely.less, the advent of using conductivity and TOC to defineRecently Boiled Water—This water may include recentlychemical purity has allowed the user to more quantitativelyor freshly boiled water (with or without mention of coolingassess the water’s chemical purity and its variability as ain the title), but cooling prior to use is clearly intended.function of routine pretreatment system maintenance andOccasionally it is necessary to use when hot. Recently Boiledregeneration. Even the presence of such unit operations asWater is specified because it is used in a pH-related test orheat exchangers and use point hoses can compromise thecarbonate-sensitive reagent, in an oxygen-sensitive test orchemical quality of water within and delivered from an oth-reagent, or in a test where outgassing could interfere witherwise well-controlled water system. Therefore, an assess-the analysis, such as specific gravity or an appearance test.ment of the consistency of the water’s chemical purity overOxygen-Free Water—The preparation of this water is time must be part of the validation program. However, evennot specifically described in the compendia. Neither is there with the most well controlled chemical quality, it is oftenan oxygen specification or analysis mentioned. However, all more difficult to consistently meet established microbiologi-uses involve analyses of materials that could be sensitive to cal quality criteria owing to phenomena occurring duringoxidation by atmospheric oxygen. Procedures for the re- and after chemical purification. A typical program involvesmoval of dissolved oxygen from solvents, though not neces- intensive daily sampling and testing of major process pointssarily water, are mentioned in Polarography ⟨801⟩ and Spec- for at least one month after operational criteria have beentrophotometry and Light-Scattering ⟨851⟩. These procedures established for each unit operation, point of use, and sam-involve simple sparging of the liquid with an inert gas such pling point.as nitrogen or helium followed by inert gas blanketing to An overlooked aspect of water system validation is theprevent oxygen reabsorption. The sparging times cited delivery of the water to its actual location of use. If thisrange from 5 to 15 minutes to an unspecified period. Some transfer process from the distribution system outlets to thePurified Water and Water for Injection systems produce water




Fig. 3. Water system validation life cycle.

water use locations (usually with hoses) is defined as outside utes of the finished water and the source water; (2) definingthe water system, then this transfer process still needs to be suitable unit operations and their operating parameters forvalidated to not adversely affect the quality of the water to achieving the desired finished water quality attributes fromthe extent it becomes unfit for use. Because routine micro- the available source water; (3) selecting piping, equipment,bial monitoring is performed for the same transfer process controls, and monitoring technologies; (4) developing an IQand components (e.g., hoses and heat exchangers) as that stage consisting of instrument calibrations, inspections toof routine water use (see Sampling Considerations), there is verify that the drawings accurately depict the final configur-some logic to include this water transfer process within the ation of the water system and, where necessary, special testsdistribution system validation. to verify that the installation meets the design requirements;

Validation is the process whereby substantiation to a high (5) developing an OQ stage consisting of tests and inspec-level of assurance that a specific process will consistently tions to verify that the equipment, system alerts, and con-produce a product conforming to an established set of qual- trols are operating reliably and that appropriate alert andity attributes is acquired and documented. Prior to and dur- action levels are established (This phase of qualification maying the very early stages of validation, the critical process overlap with aspects of the next step.); and (6) developing aparameters and their operating ranges are established. A prospective PQ stage to confirm the appropriateness of criti-validation program qualifies and documents the design, in- cal process parameter operating ranges (During this phasestallation, operation, and performance of equipment. It be- of validation, alert and action levels for key quality attributesgins when the system is defined and moves through several and operating parameters are verified.); (7) assuring the ad-stages: installation qualification (IQ), operational qualifica- equacy of ongoing control procedures, e.g., sanitization fre-tion (OQ), and performance qualification (PQ). A graphical quency; (8) supplementing a validation maintenance pro-representation of a typical water system validation life cycle gram (also called continuous validation life cycle) thatis shown in Figure 3. includes a mechanism to control changes to the water sys-

A validation plan for a water system typically includes the tem and establishes and carries out scheduled preventivefollowing steps: (1) establishing standards for quality attrib- maintenance including recalibration of instruments (In addi-




tion, validation maintenance includes a monitoring program potential problems addressed. The purpose is to highlightfor critical process parameters and a corrective action pro- issues that focus on the design, installation, operation, main-gram.); (9) instituting a schedule for periodic review of the tenance, and monitoring parameters that facilitate watersystem performance and requalification, and (10) complet- system validation.ing protocols and documenting Steps 1 through 9.

PrefiltrationPURIFIED WATER AND WATER FOR

The purpose of prefiltration—also referred to as initial,INJECTION SYSTEMScoarse, or depth filtration—is to remove solid contaminantsdown to a size of 7 to 10 µm from the incoming sourceThe design, installation, and operation of systems to pro-water supply and protect downstream system componentsduce Purified Water and Water for Injection include similarfrom particulates that can inhibit equipment performancecomponents, control techniques, and procedures. The qual-and shorten their effective life. This coarse filtration technol-ity attributes of both waters differ only in the presence of aogy utilizes primarily sieving effects for particle capture andbacterial endotoxin requirement for Water for Injection anda depth of filtration medium that has a high “dirt load”in their methods of preparation, at least at the last stage ofcapacity. Such filtration units are available in a wide rangepreparation. The similarities in the quality attributes provideof designs and for various applications. Removal efficienciesconsiderable common ground in the design of water sys-and capacities differ significantly, from granular bed filterstems to meet either requirement. The critical difference issuch as multimedia or sand for larger water systems, tothe degree of control of the system and the final purificationdepth cartridges for smaller water systems. Unit and systemsteps needed to ensure bacterial and bacterial endotoxinconfigurations vary widely in type of filtering media and lo-removal.cation in the process. Granular or cartridge prefilters areProduction of pharmaceutical water employs sequentialoften situated at or near the head of the water pretreatmentunit operations (processing steps) that address specific watersystem prior to unit operations designed to remove thequality attributes and protect the operation of subsequentsource water disinfectants. This location, however, does nottreatment steps. A typical evaluation process to select anpreclude the need for periodic microbial control because bi-appropriate water quality for a particular pharmaceuticalofilm can still proliferate, although at a slower rate in thepurpose is shown in the decision tree in Figure 2. This dia-presence of source water disinfectants. Design and opera-gram may be used to assist in defining requirements fortional issues that may impact performance of depth filtersspecific water uses and in the selection of unit operations.include channeling of the filtering media, blockage from silt,The final unit operation used to produce Water for Injectionmicrobial growth, and filtering-media loss during improperis limited to distillation or other processes equivalent or su-backwashing. Control measures involve pressure and flowperior to distillation in the removal of chemical impurities asmonitoring during use and backwashing, sanitizing, and re-well as microorganisms and their components. Distillationplacing filtering media. An important design concern is siz-has a long history of reliable performance and can be vali-ing of the filter to prevent channeling or media loss result-dated as a unit operation for the production of Water foring from inappropriate water flow rates as well as properInjection, but other technologies or combinations of technol-sizing to minimize excessively frequent or infrequent back-ogies can be validated as being equivalently effective. Otherwashing or cartridge filter replacement.technologies, such as ultrafiltration following other chemical

purification process, may be suitable in the production ofWater for Injection if they can be shown through validation Activated Carbonto be as effective and reliable as distillation. The advent ofnew materials for older technologies, such as reverse osmo- Granular activated carbon beds adsorb low molecularsis and ultrafiltration, that allow intermittent or continuous weight organic material and oxidizing additives, such asoperation at elevated, microbial temperatures, show promise chlorine and chloramine compounds, removing them fromfor a valid use in producing Water for Injection. the water. They are used to achieve certain quality attributesThe validation plan should be designed to establish the and to protect against reaction with downstream stainlesssuitability of the system and to provide a thorough under- steel surfaces, resins, and membranes. The chief operatingstanding of the purification mechanism, range of operating concerns regarding activated carbon beds include the pro-conditions, required pretreatment, and the most likely pensity to support bacteria growth, the potential for hydrau-modes of failure. It is also necessary to demonstrate the ef- lic channeling, the organic adsorption capacity, appropriatefectiveness of the monitoring scheme and to establish the water flow rates and contact time, the inability to bedocumentation and qualification requirements for the sys- regenerated in situ, and the shedding of bacteria, endotox-tem’s validation maintenance. Trials conducted in a pilot in- ins, organic chemicals, and fine carbon particles. Controlstallation can be valuable in defining the operating parame- measures may involve monitoring water flow rates and dif-ters and the expected water quality and in identifying failure ferential pressures, sanitizing with hot water or steam, back-modes. However, qualification of the specific unit operation washing, testing for adsorption capacity, and frequent re-can only be performed as part of the validation of the in- placement of the carbon bed. If the activated carbon bed isstalled operational system. The selection of specific unit op- intended for organic reduction, it may also be appropriateerations and design characteristics for a water system should to monitor influent and effluent TOC. It is important to notetake into account the quality of the feed water, the technol- that the use of steam for carbon bed sanitization is oftenogy chosen for subsequent processing steps, the extent and incompletely effective due to steam channeling rather thancomplexity of the water distribution system, and the appro- even permeation through the bed. This phenomenon canpriate compendial requirements. For example, in the design usually be avoided by using hot water sanitization. It is alsoof a system for Water for Injection, the final process (distilla- important to note that microbial biofilm development ontion or whatever other validated process is used according the surface of the granular carbon particles (as well as onto the monograph) must have effective bacterial endotoxin other particles such as found in deionizer beds and evenreduction capability and must be validated. multimedia beds) can cause adjacent bed granules to “stick”

together. When large masses of granules are agglomeratedin this fashion, normal backwashing and bed fluidizationUNIT OPERATIONS CONCERNSflow parameters may not be sufficient to disperse them,leading to ineffective removal of trapped debris, loose bi-The following is a brief description of selected unit opera-ofilm, and penetration of microbial controlling conditionstions and the operation and validation concerns associated(as well as regenerant chemicals as in the case of agglomer-with them. Not all unit operations are discussed, nor are all




ated deionizer resins). Alternative technologies to activated ness ions and possibly ammonium), and downstream filtra-carbon beds can be used in order to avoid their microbial tion to remove resin fines. If a softener is used for ammo-problems, such as disinfectant-neutralizing chemical addi- nium removal from chloramine-containing source water,tives and regenerable organic scavenging devices. However, then capacity, contact time, resin surface fouling, pH, andthese alternatives do not function by the same mechanisms regeneration frequency are very important.as activated carbon, may not be as effective at removingdisinfectants and some organics, and have a different set of

Deionizationoperating concerns and control measures that may benearly as troublesome as activated carbon beds.

Deionization (DI), and continuous electrodeionization(CEDI) are effective methods of improving the chemical

Additives quality attributes of water by removing cations and anions.DI systems have charged resins that require periodic regen-

Chemical additives are used in water systems (a) to con- eration with an acid and base. Typically, cationic resins aretrol microorganisms by use of sanitants such as chlorine regenerated with either hydrochloric or sulfuric acid, whichcompounds and ozone, (b) to enhance the removal of sus- replace the captured positive ions with hydrogen ions. An-pended solids by use of flocculating agents, (c) to remove ionic resins are regenerated with sodium or potassium hy-chlorine compounds, (d) to avoid scaling on reverse osmosis droxide, which replace captured negative ions with hydrox-membranes, and (e) to adjust pH for more effective removal ide ions. Because free endotoxin is negatively charged, thereof carbonate and ammonia compounds by reverse osmosis. is some removal of endotoxin achieved by the anionic resin.These additives do not constitute “added substances” as Both regenerant chemicals are biocidal and offer a measurelong as they are either removed by subsequent processing of microbial control. The system can be designed so thatsteps or are otherwise absent from the finished water. Con- the cation and anion resins are in separate or “twin” bedstrol of additives to ensure a continuously effective concen- or they can be mixed together to form a mixed bed. Twintration and subsequent monitoring to ensure their removal beds are easily regenerated but deionize water less effi-should be designed into the system and included in the ciently than mixed beds, which have a considerably moremonitoring program. complex regeneration process. Rechargeable resin canisters

can also be used for this purpose.The CEDI system uses a combination of mixed resin, se-

Organic Scavengers lectively permeable membranes, and an electric charge, pro-viding continuous flow (product and waste concentrate)

Organic scavenging devices use macroreticular weakly ba- and continuous regeneration. Water enters both the resinsic anion-exchange resins capable of removing organic ma- section and the waste (concentrate) section. As it passesterial and endotoxins from the water. They can be regener- through the resin, it is deionized to become product water.ated with appropriate biocidal caustic brine solutions. The resin acts as a conductor enabling the electrical poten-Operating concerns are associated with organic scavenging tial to drive the captured cations and anions through thecapacity, particulate, chemical and microbiological fouling resin and appropriate membranes for concentration and re-of the reactive resin surface, flow rate, regeneration fre- moval in the waste water stream. The electrical potentialquency, and shedding of resin fragments. Control measures also separates the water in the resin (product) section intoinclude TOC testing of influent and effluent, backwashing, hydrogen and hydroxide ions. This permits continuous re-monitoring hydraulic performance, and using downstream generation of the resin without the need for regenerant ad-filters to remove resin fines. ditives. However, unlike conventional deionization, CEDI

units must start with water that is already partially purifiedbecause they generally cannot produce Purified Water qualitySofteners when starting with the heavier ion load of unpurified sourcewater.Water softeners may be located either upstream or down- Concerns for all forms of deionization units include micro-stream of disinfectant removal units. They utilize sodium- bial and endotoxin control, chemical additive impact onbased cation-exchange resins to remove water-hardness resins and membranes, and loss, degradation, and fouling ofions, such as calcium and magnesium, that could foul or resin. Issues of concern specific to DI units include regenera-interfere with the performance of downstream processing tion frequency and completeness, channeling caused by bi-equipment such as reverse osmosis membranes, deionization ofilm agglomeration of resin particles, organic leaching fromdevices, and distillation units. Water softeners can also be new resins, complete resin separation for mixed bed regen-used to remove other lower affinity cations, such as the am- eration, and mixing air contamination (mixed beds). Controlmonium ion, that may be released from chloramine disinfec- measures vary but typically include recirculation loops, efflu-tants commonly used in drinking water and which might ent microbial control by UV light, conductivity monitoring,otherwise carryover through other downstream unit opera- resin testing, microporous filtration of mixing air, microbialtions. If ammonium removal is one of its purposes, the sof- monitoring, frequent regeneration to minimize and controltener must be located downstream of the disinfectant re- microorganism growth, sizing the equipment for suitablemoval operation, which itself may liberate ammonium from water flow and contact time, and use of elevated tempera-neutralized chloramine disinfectants. Water softener resin tures. Internal distributor and regeneration piping for mixedbeds are regenerated with concentrated sodium chloride so- bed units should be configured to ensure that regenerationlution (brine). Concerns include microorganism proliferation, chemicals contact all internal bed and piping surfaces andchanneling caused by biofilm agglomeration of resin parti- resins. Rechargeable canisters can be the source of contami-cles, appropriate water flow rates and contact time, ion-ex- nation and should be carefully monitored. Full knowledge ofchange capacity, organic and particulate resin fouling, or- previous resin use, minimum storage time between regener-ganic leaching from new resins, fracture of the resin beads, ation and use, and appropriate sanitizing procedures areresin degradation by excessively chlorinated water, and con- critical factors ensuring proper performance.tamination from the brine solution used for regeneration.

Control measures involve recirculation of water during peri-ods of low water use, periodic sanitization of the resin and Reverse Osmosisbrine system, use of microbial control devices (e.g., UV lightand chlorine), locating the unit upstream of the disinfectant Reverse osmosis (RO) units employ semipermeable mem-removal step (if used only for softening), appropriate regen- branes. The “pores” of RO membranes are actually interseg-eration frequency, effluent chemical monitoring (e.g., hard- mental spaces among the polymer molecules. They are big




enough for permeation of water molecules, but too small to ratings in the range of 10,000 to 20,000 Da are typicallypermit passage of hydrated chemical ions. However, many used in water systems for removing endotoxins. This tech-factors including pH, temperature, and differential pressure nology may be appropriate as an intermediate or final purifi-across the membrane affect the selectivity of this perme- cation step. Similar to RO, successful performance is depen-ation. With the proper controls, RO membranes can achieve dent upon pretreatment of the water by upstream unitchemical, microbial, and endotoxin quality improvement. operations.The process streams consist of supply water, product water Issues of concern for ultrafilters include compatibility of(permeate), and wastewater (reject). Depending on source membrane material with heat and sanitizing agents, mem-water, pretreatment and system configuration variations and brane integrity, fouling by particles and microorganisms,chemical additives may be necessary to achieve desired per- and seal integrity. Control measures involve filtration me-formance and reliability. dium selection, sanitization, flow design (dead end vs. tan-

A major factor affecting RO performance is the permeate gential), integrity challenges, regular cartridge changes, ele-recovery rate, that is, the amount of the water passing vated feed water temperature, and monitoring TOC andthrough the membrane compared to the amount rejected. differential pressure. Additional flexibility in operation is pos-This is influenced by the several factors, but most signifi- sible based on the way ultrafiltration units are arranged suchcantly by the pump pressure. Recoveries of 75% are typical, as in a parallel or series configurations. Care should be takenand can accomplish a 1 to 2 log purification of most impu- to avoid stagnant water conditions that could promote mi-rities. For most feed waters, this is usually not enough to croorganism growth in back-up or standby units.meet Purified Water conductivity specifications. A secondpass of this permeate water through another RO stage usu-

Charge-Modified Filtrationally achieves the necessary permeate purity if other factorssuch as pH and temperature have been appropriately ad-

Charge-modified filters are usually microbially retentive fil-justed and the ammonia from chloraminated source waterters that are treated during their manufacture to have a pos-has been previously removed. Increasing recoveries withitive charge on their surfaces. Microbial retentive filtrationhigher pressures in order to reduce the volume of rejectwill be described in a subsequent section, but the significantwater will lead to reduced permeate purity. If increasedfeature of these membranes is their electrostatic surfacepressures are needed over time to achieve the same perme-charge. Such charged filters can reduce endotoxin levels inate flow, this is an indication of partial membrane blockagethe fluids passing through them by their adsorption (owingthat needs to be corrected before it becomes irreversiblyto endotoxin’s negative charge) onto the membrane sur-fouled, and expensive membrane replacement is the onlyfaces. Though ultrafilters are more often employed as a unitoption.operation for endotoxin removal in water systems, charge-Other concerns associated with the design and operationmodified filters may also have a place in endotoxin removalof RO units include membrane materials that are extremelyparticularly where available upstream pressures are not suffi-sensitive to sanitizing agents and to particulate, chemical,cient for ultrafiltration and for a single, relatively short termand microbial membrane fouling; membrane and seal integ-use. Charge-modified filters may be difficult to validate forrity; the passage of dissolved gases, such as carbon dioxidelong-term or large-volume endotoxin retention. Evenand ammonia; and the volume of wastewater, particularlythough their purified standard endotoxin retention can bewhere water discharge is tightly regulated by local authori-well characterized, their retention capacity for “natural” en-ties. Failure of membrane or seal integrity will result in prod-dotoxins is difficult to gauge. Nevertheless, utility could beuct water contamination. Methods of control involve suita-demonstrated and validated as short-term, single-use filtersble pretreatment of the influent water stream, appropriateat points of use in water systems that are not designed formembrane material selection, integrity challenges, mem-endotoxin control or where only an endotoxin “polishing”brane design and heat tolerance, periodic sanitization, and(removal of only slight or occasional endotoxin levels) ismonitoring of differential pressures, conductivity, microbialneeded. Control and validation concerns include volumelevels, and TOC.and duration of use, flow rate, water conductivity and pu-The development of RO units that can tolerate sanitizingrity, and constancy and concentration of endotoxin levelswater temperatures as well as operate efficiently and contin-being removed. All of these factors may have to be evalu-uously at elevated temperatures has added greatly to theirated and challenged prior to using this approach, makingmicrobial control and to the avoidance of biofouling. ROthis a difficult-to-validate application. Even so, there may stillunits can be used alone or in combination with DI and CEDIbe a possible need for additional backup endotoxin testingunits as well as ultrafiltration for operational and qualityboth upstream and downstream of the filter.enhancements.

Microbial-Retentive FiltrationUltrafiltrationMicrobial-retentive membrane filters have experienced anUltrafiltration is a technology most often employed in

evolution of understanding in the past decade that haspharmaceutical water systems for removing endotoxins fromcaused previously held theoretical retention mechanisms toa water stream. It can also use semipermeable membranes,be reconsidered. These filters have a larger effective “porebut unlike RO, these typically use polysulfone membranessize” than ultrafilters and are intended to prevent the pas-whose intersegmental “pores” have been purposefully exag-sage of microorganisms and similarly sized particles withoutgerated during their manufacture by preventing the poly-unduly restricting flow. This type of filtration is widely em-mer molecules from reaching their smaller equilibrium prox-ployed within water systems for filtering the bacteria out ofimities to each other. Depending on the level of equilibriumboth water and compressed gases as well as for vent filterscontrol during their fabrication, membranes with differingon tanks and stills and other unit operations. However, themolecular weight “cutoffs” can be created such that mole-properties of the water system microorganisms seem tocules with molecular weights above these cutoffs ratings arechallenge a filter’s microbial retention from water with phe-rejected and cannot penetrate the filtration matrix.nomena absent from other aseptic filtration applications,Ceramic ultrafilters are another molecular sieving technol-such as filter sterilizing of pharmaceutical formulations priorogy. Ceramic ultrafilters are self supporting and extremelyto packaging. In the latter application, sterilizing grade fil-durable, backwashable, chemically cleanable, and steamters are generally considered to have an assigned rating ofsterilizable. However, they may require higher operating0.2 or 0.22 µm. This rather arbitrary rating is associatedpressures than membrane type ultrafilters.with filters that have the ability to retain a high level chal-All ultrafiltration devices work primarily by a molecularlenge of a specially prepared inoculum of Brevundimonassieving principle. Ultrafilters with molecular weight cutoff




(formerly Pseudomonas) diminuta. This is a small microorga- brane surface is typically hydrophobic (non-wettable bynism originally isolated decades ago from a product that water). A significant area of concern for gas filtration ishad been “filter sterilized” using a 0.45-µm rated filter. Fur- blockage of tank vents by condensed water vapor, whichther study revealed that a percentage of cells of this micro- can cause mechanical damage to the tank. Control meas-organism could reproducibly penetrate the 0.45-µm steril- ures include electrical or steam tracing and a self-drainingizing filters. Through historic correlation of B. diminuta orientation of vent filter housings to prevent accumulationretaining tighter filters, thought to be twice as good as of vapor condensate. However, a continuously high filter0.45-µm filter, assigned ratings of 0.2 or 0.22 µm with their temperature will take an oxidative toll on polypropylenesuccessful use in product solution filter sterilization, both this components of the filter, so sterilization of the unit prior tofilter rating and the associated high level B. diminuta chal- initial use, and periodically thereafter, as well as regular vis-lenge have become the current benchmarks for sterilizing ual inspections, integrity tests, and changes are recom-filtration. New evidence now suggests that for microbial- mended control methods.retentive filters used for pharmaceutical water, B. diminuta In water applications, microbial retentive filters may bemay not be the best model microorganism. used downstream of unit operations that tend to release

An archaic understanding of microbial retentive filtration microorganisms or upstream of unit operations that are sen-would lead one to equate a filter’s rating with the false im- sitive to microorganisms. Microbial retentive filters may alsopression of a simple sieve or screen that absolutely retains be used to filter water feeding the distribution system. Itparticles sized at or above the filter’s rating. A current un- should be noted that regulatory authorities allow the use ofderstanding of the mechanisms involved in microbial reten- microbial retentive filters within distribution systems or evention and the variables that can affect those mechanisms has at use points if they have been properly validated and areyielded a far more complex interaction of phenomena than appropriately maintained. A point-of-use filter should onlypreviously understood. A combination of simple sieve reten- be intended to “polish” the microbial quality of an other-tion and surface adsorption are now known to contribute to wise well-maintained system and not to serve as the primarymicrobial retention. microbial control device. The efficacy of system microbial

The following all interact to create some unusual and sur- control measures can only be assessed by sampling theprising retention phenomena for water system microorgan- water upstream of the filters. As an added measure of pro-isms: the variability in the range and average pore sizes cre- tection, in-line UV lamps, appropriately sized for the flowated by the various membrane fabrication processes, the rate (see Sanitization), may be used just upstream of micro-variability of the surface chemistry and three-dimensional bial retentive filters to inactivate microorganisms prior tostructure related to the different polymers used in these fil- their capture by the filter. This tandem approach tends toter matrices, and the size and surface properties of the mi- greatly delay potential microbial penetration phenomenacroorganism intended to be retained by the filters. B. and can substantially extend filter service life.diminuta may not be the best challenge microorganisms fordemonstrating bacterial retention for 0.2- to 0.22-µm rated

Ultraviolet Lightfilters for use in water systems because it appears to bemore easily retained by these filters than some water system

The use of low-pressure UV lights that emit a 254-nmflora. The well-documented appearance of water system mi-wavelength for microbial control is discussed under Sanitiza-croorganisms on the downstream sides of some 0.2- totion, but the application of UV light in chemical purification0.22-µm rated filters after a relatively short period of useis also emerging. This 254-nm wavelength is also useful inseems to support that some penetration phenomena are atthe destruction of ozone. With intense emissions at wave-work. Unknown for certain is if this downstream appearancelengths around 185 nm (as well as at 254 nm), mediumis caused by a “blow-through” or some other pass-throughpressure UV lights have demonstrated utility in the destruc-phenomenon as a result of tiny cells or less cell “stickiness”,tion of the chlorine-containing disinfectants used in sourceor by a “growth through” phenomenon as a result of cellswater as well as for interim stages of water pretreatment.hypothetically replicating their way through the pores to theHigh intensities of this wavelength alone or in combinationdownstream side. Whatever is the penetration mechanism,with other oxidizing sanitants, such as hydrogen peroxide,0.2- to 0.22-µm rated membranes may not be the besthave been used to lower TOC levels in recirculating distribu-choice for some water system uses.tion systems. The organics are typically converted to carbonMicrobial retention success in water systems has been re-dioxide, which equilibrates to bicarbonate, and incompletelyported with the use of some manufacturers’ filters arbitrarilyoxidized carboxylic acids, both of which can easily be re-rated as 0.1 µm. There is general agreement that for a givenmoved by polishing ion-exchange resins. Areas of concernmanufacturer, their 0.1-µm rated filters are tighter than theirinclude adequate UV intensity and residence time, gradual0.2- to 0.22-µm rated filters. However, comparably ratedloss of UV emissivity with bulb age, gradual formation offilters from different manufacturers in water filtration appli-UV-absorbing film at the water contact surface, incompletecations may not perform equivalently owing to the differentphotodegradation during unforeseen source water hyper-filter fabrication processes and the nonstandardized micro-chlorination, release of ammonia from chloraminebial retention challenge processes currently used for definingphotodegradation, unapparent UV bulb failure, and conduc-the 0.1-µm filter rating. It should be noted that use of 0.1-tivity degradation in distribution systems using 185-nm UVµm rated membranes generally results in a sacrifice in flowlights. Control measures include regular inspection or emis-rate compared to 0.2- to 0.22-µm membranes, so whateversivity alarms to detect bulb failures or film occlusions, regu-membranes are chosen for a water system application, thelar UV bulb sleeve cleaning and wiping, downstream chlo-user must verify that the membranes are suitable for theirrine detectors, downstream polishing deionizers, and regularintended application, use period, and use process, including(approximately yearly) bulb replacement.flow rate.

For microbial retentive gas filtrations, the same sievingand adsorptive retention phenomena are at work as in liq- Distillationuid filtration, but the adsorptive phenomenon is enhancedby additional electrostatic interactions between particles and Distillation units provide chemical and microbial purifica-filter matrix. These electrostatic interactions are so strong tion via thermal vaporization, mist elimination, and waterthat particle retention for a given filter rating is significantly vapor condensation. A variety of designs is available includ-more efficient in gas filtration than in water or product solu- ing single effect, multiple effect, and vapor compression.tion filtrations. These additional adsorptive interactions The latter two configurations are normally used in largerrender filters rated at 0.2 to 0.22 µm unquestionably suita- systems because of their generating capacity and efficiency.ble for microbial retentive gas filtrations. When microbially Distilled water systems require different feed water controlsretentive filters are used in these applications, the mem-




than required by membrane systems. For distillation, due tain. Pumps should be designed to deliver fully turbulentconsideration must be given to prior removal of hardness flow conditions to facilitate thorough heat distribution (forand silica impurities that may foul or corrode the heat trans- hot water sanitized systems) as well as thorough chemicalfer surfaces as well as prior removal of those impurities that sanitant distribution. Turbulent flow also appear to eithercould volatize and condense along with the water vapor. In retard the development of biofilms or reduce the tendencyspite of general perceptions, even the best distillation pro- of those biofilms to shed bacteria into the water. If redun-cess cannot afford absolute removal of contaminating ions dant pumps are used, they should be configured and usedand endotoxin. Most stills are recognized as being able to to avoid microbial contamination of the system.accomplish at least a 3 to 4 log reduction in these impurity Components and distribution lines should be sloped andconcentrations. Areas of concern include carry-over of vola- fitted with drain points so that the system can be com-tile organic impurities such as trihalomethanes (see Source pletely drained. In stainless steel distribution systems whereand Feed Water Considerations) and gaseous impurities such the water is circulated at a high temperature, dead legs andas ammonia and carbon dioxide, faulty mist elimination, low-flow conditions should be avoided, and valved tie-inevaporator flooding, inadequate blowdown, stagnant water points should have length-to-diameter ratios of six or less. Ifin condensers and evaporators, pump and compressor seal constructed of heat tolerant plastic, this ratio should bedesign, pinhole evaporator and condenser leaks, and con- even less to avoid cool points where biofilm developmentductivity (quality) variations during start-up and operation. could occur. In ambient temperature distribution systems,

Methods of control may involve preliminary decarbona- particular care should be exercised to avoid or minimizetion steps to remove both dissolved carbon dioxide and dead leg ratios of any size and provide for complete drain-other volatile or noncondensable impurities; reliable mist age. If the system is intended to be steam sanitized, carefulelimination to minimize feedwater droplet entrainment; vis- sloping and low-point drainage is crucial to condensate re-ual or automated high water level indication to detect boiler moval and sanitization success. If drainage of componentsflooding and boil over; use of sanitary pumps and compres- or distribution lines is intended as a microbial control strat-sors to minimize microbial and lubricant contamination of egy, they should also be configured to be completely driedfeedwater and condensate; proper drainage during inactive using dry compressed air (or nitrogen if appropriate em-periods to minimize microbial growth and accumulation of ployee safety measures are used). Drained but still moist sur-associated endotoxin in boiler water; blow down control to faces will still support microbial proliferation. Water exitinglimit the impurity concentration effect in the boiler to man- from the distribution system should not be returned to theageable levels; on-line conductivity sensing with automated system without first passing through all or a portion of thediversion to waste to prevent unacceptable water upon still purification train.startup or still malfunction from getting into the finished The distribution design should include the placement ofwater distribute system; and periodic integrity testing for sampling valves in the storage tank and at other locations,pinhole leaks to routinely assure condensate is not compro- such as in the return line of the recirculating water system.mised by nonvolatized source water contaminants. Where feasible, the primary sampling sites for water should

be the valves that deliver water to the points of use. Directconnections to processes or auxiliary equipment should be

Storage Tanks designed to prevent reverse flow into the controlled watersystem. Hoses and heat exchangers that are attached to

Storage tanks are included in water distribution systems points of use in order to deliver water for a particular useto optimize processing equipment capacity. Storage also al- must not chemically or microbiologically degrade the waterlows for routine maintenance within the pretreatment train quality. The distribution system should permit sanitizationwhile maintaining continuous supply to meet manufacturing for microorganism control. The system may be continuouslyneeds. Design and operation considerations are needed to operated at sanitizing conditions or sanitized periodically.prevent or minimize the development of biofilm, to mini-mize corrosion, to aid in the use of chemical sanitization ofthe tanks, and to safeguard mechanical integrity. These con- INSTALLATION, MATERIALS OFsiderations may include using closed tanks with smooth in- CONSTRUCTION, AND COMPONENTteriors, the ability to spray the tank headspace using SELECTIONsprayballs on recirculating loop returns, and the use ofheated, jacketed/insulated tanks. This minimizes corrosion Installation techniques are important because they can af-and biofilm development and aids in thermal and chemical fect the mechanical, corrosive, and sanitary integrity of thesanitization. Storage tanks require venting to compensate system. Valve installation attitude should promote gravityfor the dynamics of changing water levels. This can be ac- drainage. Pipe supports should provide appropriate slopescomplished with a properly oriented and heat-traced filter for drainage and should be designed to support the pipinghousing fitted with a hydrophobic microbial retentive mem- adequately under worst-case thermal and flow conditions.brane filter affixed to an atmospheric vent. Alternatively, an The methods of connecting system components includingautomatic membrane-filtered compressed gas blanketing units of operation, tanks, and distribution piping requiresystem may be used. In both cases, rupture disks equipped careful attention to preclude potential problems. Stainlesswith a rupture alarm device should be used as a further steel welds should provide reliable joints that are internallysafeguard for the mechanical integrity of the tank. Areas of smooth and corrosion-free. Low-carbon stainless steel, com-concern include microbial growth or corrosion due to irreg- patible wire filler, where necessary, inert gas, automaticular or incomplete sanitization and microbial contamination welding machines, and regular inspection and documenta-from unalarmed rupture disk failures caused by condensate- tion help to ensure acceptable weld quality. Follow-upoccluded vent filters. cleaning and passivation are important for removing con-

tamination and corrosion products and to re-establish thepassive corrosion resistant surface. Plastic materials can beDistribution Systemsfused (welded) in some cases and also require smooth, uni-form internal surfaces. Adhesive glues and solvents shouldDistribution system configuration should allow for thebe avoided due to the potential for voids and extractables.continuous flow of water in the piping by means of recircu-Mechanical methods of joining, such as flange fittings, re-lation. Use of nonrecirculating, dead-end, or one-way sys-quire care to avoid the creation of offsets, gaps, penetra-tems or system segments should be avoided whenever pos-tions, and voids. Control measures include good alignment,sible. If not possible, these systems should be periodicallyproperly sized gaskets, appropriate spacing, uniform sealingflushed and more closely monitored. Experience has shownforce, and the avoidance of threaded fittings.that continuously recirculated systems are easier to main-




Materials of construction should be selected to be com- and may leave biofilms intact. Compounds such as hydro-patible with control measures such as sanitizing, cleaning, gen peroxide, ozone, and peracetic acid oxidize bacteriaand passivating. Temperature rating is a critical factor in and biofilms by forming reactive peroxides and free radicalschoosing appropriate materials because surfaces may be re- (notably hydroxyl radicals). The short half-life of ozone inquired to handle elevated operating and sanitization tem- particular, and its limitation on achievable concentrations re-peratures. Should chemicals or additives be used to clean, quire that it be added continuously during the sanitizationcontrol, or sanitize the system, materials resistant to these process. Hydrogen peroxide and ozone rapidly degrade tochemicals or additives must be utilized. Materials should be water and oxygen; peracetic acid degrades to acetic acid incapable of handling turbulent flow and elevated velocities the presence of UV light. In fact, ozone’s ease of degrada-without wear of the corrosion-resistant film such as the pas- tion to oxygen using 254-nm UV lights at use points allow itsive chromium oxide surface of stainless steel. The finish on to be most effectively used on a continuous basis to providemetallic materials such as stainless steel, whether it is a re- continuously sanitizing conditions.fined mill finish, polished to a specific grit, or an electropol- In-line UV light at a wavelength of 254 nm can also beished treatment, should complement system design and used to continuously “sanitize” water circulating in the sys-provide satisfactory corrosion and microbial activity resis- tem, but these devices must be properly sized for the watertance as well as chemical sanitizability. Auxiliary equipment flow. Such devices inactivate a high percentage (but notand fittings that require seals, gaskets, diaphragms, filter 100%) of microorganisms that flow through the device butmedia, and membranes should exclude materials that per- cannot be used to directly control existing biofilm upstreammit the possibility of extractables, shedding, and microbial or downstream of the device. However, when coupled withactivity. Insulating materials exposed to stainless steel sur- conventional thermal or chemical sanitization technologiesfaces should be free of chlorides to avoid the phenomenon or located immediately upstream of a microbially retentiveof stress corrosion cracking that can lead to system contami- filter, it is most effective and can prolong the interval be-nation and the destruction of tanks and critical system tween system sanitizations.components. It is important to note that microorganisms in a well-

Specifications are important to ensure proper selection of developed biofilm can be extremely difficult to kill, even bymaterials and to serve as a reference for system qualification aggressive oxidizing biocides. The less developed and there-and maintenance. Information such as mill reports for stain- fore thinner the biofilm, the more effective the biocidal ac-less steel and reports of composition, ratings, and material tion. Therefore, optimal biocide control is achieved by fre-handling capabilities for nonmetallic substances should be quent biocide use that does not allow significant biofilmreviewed for suitability and retained for reference. Compo- development between treatments.nent (auxiliary equipment) selection should be made with Sanitization steps require validation to demonstrate theassurance that it does not create a source of contamination capability of reducing and holding microbial contaminationintrusion. Heat exchangers should be constructed to prevent at acceptable levels. Validation of thermal methods shouldleakage of heat transfer medium to the pharmaceutical include a heat distribution study to demonstrate that sani-water and, for heat exchanger designs where prevention tization temperatures are achieved throughout the system,may fail, there should be a means to detect leakage. Pumps including the body of use point valves. Validation of chemi-should be of sanitary design with seals that prevent contam- cal methods require demonstrating adequate chemical con-ination of the water. Valves should have smooth internal sur- centrations throughout the system, exposure to all wettedfaces with the seat and closing device exposed to the flush- surfaces, including the body of use point valves, and com-ing action of water, such as occurs in diaphragm valves. plete removal of the sanitant from the system at the com-Valves with pocket areas or closing devices (e.g., ball, plug, pletion of treatment. Methods validation for the detectiongate, globe) that move into and out of the flow area should and quantification of residues of the sanitant or its objec-be avoided. tionable degradants is an essential part of the validation

program. The frequency of sanitization should be supportedby, if not triggered by, the results of system microbial moni-

SANITIZATION toring. Conclusions derived from trend analysis of the mi-crobiological data should be used as the alert mechanism

Microbial control in water systems is achieved primarily for maintenance. The frequency of sanitization should bethrough sanitization practices. Systems can be sanitized us- established in such a way that the system operates in a stateing either thermal or chemical means. Thermal approaches of microbiological control and does not routinely exceedto system sanitization include periodic or continuously circu- alert levels (see Alert and Action Levels and Specifications).lating hot water and the use of steam. Temperatures of atleast 80° are most commonly used for this purpose, butcontinuously recirculating water of at least 65° has also OPERATION, MAINTENANCE, AND CONTROLbeen used effectively in insulated stainless steel distributionsystems when attention is paid to uniformity and distribu- A preventive maintenance program should be establishedtion of such self-sanitizing temperatures. These techniques to ensure that the water system remains in a state of con-are limited to systems that are compatible with the higher trol. The program should include (1) procedures for operat-temperatures needed to achieve sanitization. Although ther- ing the system, (2) monitoring programs for critical qualitymal methods control biofilm development by either contin- attributes and operating conditions including calibration ofuously inhibiting their growth or, in intermittent applica- critical instruments, (3) schedule for periodic sanitization, (4)tions, by killing the microorganisms within biofilms, they are preventive maintenance of components, and (5) control ofnot effective in removing established biofilms. Killed but in- changes to the mechanical system and to operatingtact biofilms can become a nutrient source for rapid biofilm conditions.regrowth after the sanitizing conditions are removed or Operating Procedures—Procedures for operating thehalted. In such cases, a combination of routine thermal and water system and performing routine maintenance and cor-periodic supplementation with chemical sanitization might rective action should be written, and they should also definebe more effective. The more frequent the thermal sanitiza- the point when action is required. The procedures shouldtion, the more likely biofilm development and regrowth can be well documented, detail the function of each job, assignbe eliminated. Chemical methods, where compatible, can who is responsible for performing the work, and describebe used on a wider variety of construction materials. These how the job is to be conducted. The effectiveness of thesemethods typically employ oxidizing agents such as haloge- procedures should be assessed during water systemnated compounds, hydrogen peroxide, ozone, peracetic validation.acid, or combinations thereof. Halogenated compounds areeffective sanitizers but are difficult to flush from the system




Monitoring Program—Critical quality attributes and op- only indicative of the concentration of planktonic (free float-erating parameters should be documented and monitored. ing) microorganisms present in the system. Biofilm microor-The program may include a combination of in-line sensors ganisms (those attached to water system surfaces) are usu-or automated instruments (e.g., for TOC, conductivity, ally present in greater numbers and are the source of thehardness, and chlorine), automated or manual documenta- planktonic population recovered from grab samples. Micro-tion of operational parameters (such as flow rates or pres- organisms in biofilms represent a continuous source of con-sure drop across a carbon bed, filter, or RO unit), and labo- tamination and are difficult to directly sample and quantify.ratory tests (e.g., total microbial counts). The frequency of Consequently, the planktonic population is usually used assampling, the requirement for evaluating test results, and an indicator of system contamination levels and is the basisthe necessity for initiating corrective action should be for system Alert and Action Levels. The consistent appearanceincluded. of elevated planktonic levels is usually an indication of ad-

vanced biofilm development in need of remedial control.Sanitization—Depending on system design and the se-System control and sanitization are key in controlling biofilmlected units of operation, routine periodic sanitization mayformation and the consequent planktonic population.be necessary to maintain the system in a state of microbial

Sampling for chemical analyses is also done for in-processcontrol. Technologies for sanitization are described above.control and for quality control purposes. However, unlikePreventive Maintenance—A preventive maintenance microbial analyses, chemical analyses can be and often areprogram should be in effect. The program should establish performed using on-line instrumentation. Such on-line test-what preventive maintenance is to be performed, the fre- ing has unequivocal in-process control purposes because itquency of maintenance work, and how the work should be is not performed on the water delivered from the system.documented. However, unlike microbial attributes, chemical attributes are

Change Control—The mechanical configuration and op- usually not significantly degraded by hoses. Therefore,erating conditions must be controlled. Proposed changes through verification testing, it may be possible to show thatshould be evaluated for their impact on the whole system. the chemical attributes detected by the on-line instrumenta-The need to requalify the system after changes are made tion (in-process testing) are equivalent to those detected atshould be determined. Following a decision to modify a the ends of the use point hoses (quality control testing).water system, the affected drawings, manuals, and proce- This again creates a single sample and mixed data use sce-dures should be revised. nario. It is far better to operate the instrumentation in a

continuous mode, generating large volumes of in-processdata, but only using a defined small sampling of that dataSAMPLING CONSIDERATIONS for QC purposes. Examples of acceptable approaches in-clude using highest values for a given period, highest time-Water systems should be monitored at a frequency that is weighted average for a given period (from fixed or rollingsufficient to ensure that the system is in control and contin- sub-periods), or values at a fixed daily time. Each approachues to produce water of acceptable quality. Samples should has advantages and disadvantages relative to calculationbe taken from representative locations within the processing complexity and reflection of continuous quality, so the userand distribution system. Established sampling frequencies must decide which approach is most suitable or justifiable.should be based on system validation data and should cover

critical areas including unit operation sites. The samplingplan should take into consideration the desired attributes of CHEMICAL CONSIDERATIONSthe water being sampled. For example, systems for Water forInjection because of their more critical microbiological re- The chemical attributes of Purified Water and Water forquirements, may require a more rigorous sampling Injection in effect prior to USP 23 were specified by a seriesfrequency. of chemistry tests for various specific and nonspecific attrib-

Analyses of water samples often serve two purposes: in- utes with the intent of detecting chemical species indicativeprocess control assessments and final quality control assess- of incomplete or inadequate purification. While these meth-ments. In-process control analyses are usually focused on ods could have been considered barely adequate to controlthe attributes of the water within the system. Quality con- the quality of these waters, they nevertheless stood the testtrol is primarily concerned with the attributes of the water of time. This was partly because the operation of water sys-delivered by the system to its various uses. The latter usually tems was, and still is, based on on-line conductivity meas-employs some sort of transfer device, often a flexible hose, urements and specifications generally thought to precludeto bridge the gap between the distribution system use-point the failure of these archaic chemistry attribute tests.valve and the actual location of water use. The issue of sam- USP moved away from these chemical attribute tests tople collection location and sampling procedure is often contemporary analytical technologies for the bulk waters Pu-hotly debated because of the typically mixed use of the data rified Water and Water for Injection. The intent was to up-generated from the samples, for both in-process control and grade the analytical technologies without tightening thequality control. In these single sample and mixed data use quality requirements. The two contemporary analytical tech-situations, the worst-case scenario should be utilized. In nologies employed were TOC and conductivity. The TOCother words, samples should be collected from use points test replaced the test for Oxidizable substances that primarilyusing the same delivery devices, such as hoses, and proce- targeted organic contaminants. A multistaged Conductivitydures, such as preliminary hose or outlet flushing, as are test which detects ionic (mostly inorganic) contaminants re-employed by production from those use points. Where use placed, with the exception of the test for Heavy metals, allpoints per se cannot be sampled, such as hard-piped con- of the inorganic chemical tests (i.e., Ammonia, Calcium, Car-nections to equipment, special sampling ports may be used. bon dioxide, Chloride, Sulfate).In all cases, the sample must represent as closely as possible Replacing the heavy metals attribute was considered un-the quality of the water used in production. If a point of use necessary because (a) the source water specifications (foundfilter is employed, sampling of the water prior to and after in the NPDWR) for individual Heavy metals were tighter thanthe filter is needed because the filter will mask the microbial the approximate limit of detection of the Heavy metals testcontrol achieved by the normal operating procedures of the for USP XXII Water for Injection and Purified Water (approxi-system. mately 0.1 ppm), (b) contemporary water system construc-

Samples containing chemical sanitizing agents require tion materials do not leach heavy metal contaminants, andneutralization prior to microbiological analysis. Samples for (c) test results for this attribute have uniformly been nega-microbiological analysis should be tested immediately, or tive—there has not been a confirmed occurrence of a singu-suitably refrigerated to preserve the original microbial attrib- lar test failure (failure of only the Heavy metals test with allutes until analysis can begin. Samples of flowing water are other attributes passing) since the current heavy metal




Table 1. Contributing Ion Conductivities of the Chloride–Ammonia Model as a Function of pH(in atmosphere-equilibrated water at 25°)

Conductivity(µS/cm)

Combined Stage 3pH H+ OH– HCO3– Cl– Na+ NH4+ Conductivities Limit5.0 3.49 0 0.02 1.01 0.19 0 4.71 4.75.1 2.77 0 0.02 1.01 0.29 0 4.09 4.15.2 2.20 0 0.03 1.01 0.38 0 3.62 3.65.3 1.75 0 0.04 1.01 0.46 0 3.26 3.35.4 1.39 0 0.05 1.01 0.52 0 2.97 3.05.5 1.10 0 0.06 1.01 0.58 0 2.75 2.85.6 0.88 0 0.08 1.01 0.63 0 2.60 2.65.7 0.70 0 0.10 1.01 0.68 0 2.49 2.55.8 0.55 0 0.12 1.01 0.73 0 2.41 2.45.9 0.44 0 0.16 1.01 0.78 0 2.39 2.46.0 0.35 0 0.20 1.01 0.84 0 2.40 2.46.1 0.28 0 0.25 1.01 0.90 0 2.44 2.46.2 0.22 0 0.31 1.01 0.99 0 2.53 2.56.3 0.18 0 0.39 0.63 0 1.22 2.42 2.46.4 0.14 0.01 0.49 0.45 0 1.22 2.31 2.36.5 0.11 0.01 0.62 0.22 0 1.22 2.18 2.26.6 0.09 0.01 0.78 0 0.04 1.22 2.14 2.16.7 0.07 0.01 0.99 0 0.27 1.22 2.56 2.66.8 0.06 0.01 1.24 0 0.56 1.22 3.09 3.16.9 0.04 0.02 1.56 0 0.93 1.22 3.77 3.87.0 0.03 0.02 1.97 0 1.39 1.22 4.63 4.6

drinking water standards have been in place. Nevertheless, pending on the pH-induced ionic imbalance (see Table 1).since the presence of heavy metals in Purified Water or Water The Stage 2 conductivity specification is the lowest value onfor Injection could have dire consequences, its absence this table, 2.1 µS/cm. The Stage 1 specifications, designedshould at least be documented during new water system primarily for on-line measurements, were derived essentiallycommissioning and validation or through prior test results by summing the lowest values in the contributing ion col-records. umns for each of a series of tables similar to Table 1, created

Total solids and pH were the only tests not covered by for each 5° increment between 0° and 100°. For exampleconductivity testing. The test for Total solids was considered purposes, the italicized values in Table 1, the conductivityredundant because the nonselective tests of conductivity data table for 25°, were summed to yield a conservativeand TOC could detect most chemical species other than value of 1.3 µS/cm, the Stage 1 specification for asilica, which could remain undetected in its colloidal form. nontemperature compensated, nonatmosphere equilibratedColloidal silica in Purified Water and Water for Injection is water sample that actually had a measured temperature ofeasily removed by most water pretreatment steps and even 25° to 29°. Each 5° increment in the table was similarlyif present in the water, constitutes no medical or functional treated to yield the individual values listed in the table ofhazard except under extreme and rare situations. In such Stage 1 specifications (see Water Conductivity, Bulk Waterextreme situations, other attribute extremes are also likely to ⟨645⟩).be detected. It is, however, the user’s responsibility to en- As stated above, this rather radical change to utilizing asure fitness for use. If silica is a significant component in the conductivity attribute as well as the inclusion of a TOC attri-source water, and the purification unit operations could be bute allowed for on-line measurements. This was a majoroperated or fail and selectively allow silica to be released philosophical change and allowed major savings to be real-into the finished water (in the absence of co- ized by industry. The TOC and conductivity tests can alsocontaminants detectable by conductivity), then either silica- be performed “off-line” in the laboratories using collectedspecific or a total solids type testing should be utilized to samples, though sample collection tends to introduce op-monitor and control this rare problem. portunities for adventitious contamination that can cause

The pH attribute was eventually recognized to be redun- false high readings. The collection of on-line data is not,dant to the conductivity test (which included pH as an as- however, without challenges. The continuous readings tendpect of the test and specification); therefore, pH was to create voluminous amounts of data where before only adropped as a separate attribute test. single data point was available. As stated under Sampling

The rationale used by USP to establish its Purified Water Considerations, continuous in-process data is excellent forand Water for Injection conductivity specifications took into understanding how a water system performs during all of itsconsideration the conductivity contributed by the two least various usage and maintenance events in real time, but isconductive former attributes of Chloride and Ammonia, too much data for QC purposes. Therefore, a justifiable frac-thereby precluding their failure had those wet chemistry tion or averaging of the data can be used that is still repre-tests been performed. In essence, the Stage 3 conductivity sentative of the overall water quality being used.specifications (see Water Conductivity, Bulk Water ⟨645⟩) Packaged/sterile waters present a particular dilemma rela-were established from the sum of the conductivities of the tive to the attributes of conductivity and TOC. The packagelimit concentrations of chloride ions (from pH 5.0 to 6.2) itself is the source of chemicals (inorganics and organics)and ammonia ions (from pH 6.3 to 7.0), plus the unavoida- that leach over time into the water and can easily be de-ble contribution of other conductivity-contributing ions from tected. The irony of organic leaching from plastic packagingwater (H+ and OH–), dissolved atmospheric CO2 (as HCO3–), is that when the Oxidizable substances test was the only “or-and an electro-balancing quantity of either Na+ or Cl–, de- ganic contaminant” test for both bulk and packaged/sterile




waters, that test’s insensitivity to those organic leachables Unit operations can be a major source of endogenous mi-allowed their presence in packaged/sterile water to be quite crobial contamination. Microorganisms present in feedhigh (possibly many times the TOC specification for bulk water may adsorb to carbon bed, deionizer resins, filterwater). Similarly, glass containers can also leach inorganics, membranes, and other unit operation surfaces and initiatesuch as sodium, which are easily detected by conductivity, the formation of a biofilm. In a high-purity water system,but poorly detected by the former wet chemistry attribute biofilm is an adaptive response by certain microorganisms totests. Most of these leachables are considered harmless by survive in this low nutrient environment. Downstream colo-current perceptions and standards at the rather significant nization can occur when microorganisms are shed from ex-concentrations present. Nevertheless, they effectively de- isting biofilm-colonized surfaces and carried to other areasgrade the quality of the high-purity waters placed into these of the water system. Microorganisms may also attach to sus-packaging systems. Some packaging materials contain more pended particles such as carbon bed fines or fractured resinleachables than others and may not be as suitable for hold- particles. When the microorganisms become planktonic,ing water and maintaining its purity. they serve as a source of contamination to subsequent puri-

The attributes of conductivity and TOC tend to reveal fication equipment (compromising its functionality) and tomore about the packaging leachables than they do about distribution systems.the water’s original purity. These currently “allowed” leach- Another source of endogenous microbial contamination isables could render the packaged/sterile versions of originally the distribution system itself. Microorganisms can colonizeequivalent bulk water essentially unsuitable for many uses pipe surfaces, rough welds, badly aligned flanges, valves,where the bulk waters are perfectly adequate. and unidentified dead legs, where they proliferate, forming

Therefore, to better control the ionic packaging leach- a biofilm. The smoothness and composition of the surfaceables, Water Conductivity ⟨645⟩ is divided into two sections. may affect the rate of initial microbial adsorption, but onceThe first is titled Bulk Water, which applies to Purified Water, adsorbed, biofilm development, unless otherwise inhibitedWater for Injection, Water for Hemodialysis, and Pure Steam, by sanitizing conditions, will occur regardless of the surface.and includes the three-stage conductivity testing instructions Once formed, the biofilm becomes a continuous source ofand specifications. The second is titled Sterile Water, which microbial contamination.applies to Sterile Purified Water, Sterile Water for Injection,Sterile Water for Inhalation, and Sterile Water for Irrigation.

ENDOTOXIN CONSIDERATIONSThe Sterile Water section includes conductivity specificationssimilar to the Stage 2 testing approach because it is in-

Endotoxins are lipopolysaccharides found in and shedtended as a laboratory test, and these sterile waters werefrom the cell envelope that is external to the cell wall ofmade from bulk water that already complied with the three-Gram-negative bacteria. Gram-negative bacteria that formstage conductivity test. In essence, packaging leachablesbiofilms can become a source of endotoxins in pharmaceuti-are the primary target “analytes” of the conductivity specifi-cal waters. Endotoxins may occur as clusters of lipo-cations in the Sterile Water section of Water Conductivitypolysaccharide molecules associated with living microorgan-⟨645⟩. The effect on potential leachables from different con-isms, fragments of dead microorganisms or thetainer sizes is the rationale for having two different specifica-polysaccharide slime surrounding biofilm bacteria, or as freetions, one for small packages containing nominal volumes ofmolecules. The free form of endotoxins may be released10 mL or less and another for larger packages. These con-from cell surfaces of the bacteria that colonize the waterductivity specifications are harmonized with the Europeansystem, or from the feed water that may enter the waterPharmacopoeia conductivity specifications for Sterile Watersystem. Because of the multiplicity of endotoxin sources in afor Injection. All monographed waters, except Bacteriostaticwater system, endotoxin quantitation in a water system isWater for Injection, have a conductivity specification that di-not a good indicator of the level of biofilm abundancerects the user to either the Bulk Water or the Sterile Waterwithin a water system.section of Water Conductivity ⟨645⟩. For the sterile water

Endotoxin levels may be minimized by controlling the in-monographs, this water conductivity specification replacestroduction of free endotoxins and microorganisms in thethe redundant wet chemistry limit tests intended for inor-feed water and minimizing microbial proliferation in the sys-ganic contaminants that had previously been specified intem. This may be accomplished through the normal exclu-these monographs.sion or removal action afforded by various unit operationswithin the treatment system as well as through system sani-

MICROBIAL CONSIDERATIONS tization. Other control methods include the use of ultrafiltersor charge-modified filters, either in-line or at the point of

The major exogenous source of microbial contamination use. The presence of endotoxins may be monitored as de-of bulk pharmaceutical water is source or feed water. Feed scribed in the general test chapter Bacterial Endotoxins Testwater quality must, at a minimum, meet the quality attrib- ⟨85⟩.utes of Drinking Water for which the level of coliforms areregulated. A wide variety of other microorganisms, chiefly

MICROBIAL ENUMERATIONGram-negative bacteria, may be present in the incomingwater. These microorganisms may compromise subsequent CONSIDERATIONSpurification steps. Examples of other potential exogenoussources of microbial contamination include unprotected The objective of a water system microbiological monitor-vents, faulty air filters, ruptured rupture disks, backflow from ing program is to provide sufficient information to controlcontaminated outlets, unsanitized distribution system “open- and assess the microbiological quality of the water pro-ings” including routine component replacements, inspec- duced. Product quality requirements should dictate watertions, repairs, and expansions, inadequate drain and air- quality specifications. An appropriate level of control may bebreaks, and replacement activated carbon, deionizer resins, maintained by using data trending techniques and, if neces-and regenerant chemicals. In these situations, the exoge- sary, limiting specific contraindicated microorganisms. Con-nous contaminants may not be normal aquatic bacteria but sequently, it may not be necessary to detect all of the mi-rather microorganisms of soil or even human origin. The croorganisms species present in a given sample. Thedetection of nonaquatic microorganisms may be an indica- monitoring program and methodology should indicate ad-tion of a system component failure, which should trigger verse trends and detect microorganisms that are potentiallyinvestigations that will remediate their source. Sufficient care harmful to the finished product, process, or consumer. Finalshould be given to system design and maintenance in order selection of method variables should be based on the indi-to minimize microbial contamination from these exogenous vidual requirements of the system being monitored.sources.




It should be recognized that there is no single method beneficial for isolating slow growing “oligotrophic” bacteriathat is capable of detecting all of the potential microbial and bacteria that require lower levels of nutrients to growcontaminants of a water system. The methods used for mi- optimally. Often some facultative oligotrophic bacteria arecrobial monitoring should be capable of isolating the num- able to grow on high nutrient media and some facultativebers and types of organisms that have been deemed signifi- copiotrophic bacteria are able to grow on low-nutrient me-cant relative to in-process system control and product dia, but this overlap is not complete. Low-nutrient andimpact for each individual system. Several criteria should be high-nutrient cultural approaches may be concurrently used,considered when selecting a method to monitor the micro- especially during the validation of a water system, as well asbial content of a pharmaceutical water system. These in- periodically thereafter. This concurrent testing could deter-clude method sensitivity, range of organisms types or spe- mine if any additional numbers or types of bacteria can becies recovered, sample processing throughput, incubation preferentially recovered by one of the approaches. If so, theperiod, cost, and methodological complexity. An alternative impact of these additional isolates on system control andconsideration to the use of the classical “culture” ap- the end uses of the water could be assessed. Also, the effi-proaches is a sophisticated instrumental or rapid test cacy of system controls and sanitization on these additionalmethod that may yield more timely results. However, care isolates could be assessed.must be exercised in selecting such an alternative approach Duration and temperature of incubation are also criticalto ensure that it has both sensitivity and correlation to class- aspects of a microbiological test method. Classical method-ical culture approaches, which are generally considered the ologies using high nutrient media are typically incubated ataccepted standards for microbial enumeration. 30° to 35° for 48 to 72 hours. Because of the flora in cer-

Consideration should also be given to the timeliness of tain water systems, incubation at lower temperatures (e.g.,microbial enumeration testing after sample collection. The 20° to 25°) for longer periods (e.g., 5 to 7 days) can re-number of detectable planktonic bacteria in a sample col- cover higher microbial counts when compared to classicallected in a scrupulously clean sample container will usually methods. Low-nutrient media are designed for these lowerdrop as time passes. The planktonic bacteria within the sam- temperature and longer incubation conditions (sometimesple will tend to either die or to irretrievably adsorb to the as long as 14 days to maximize recovery of very slow grow-container walls reducing the number of viable planktonic ing oligotrophs or sanitant injured microorganisms), butbacteria that can be withdrawn from the sample for testing. even high-nutrient media can sometimes increase their re-The opposite effect can also occur if the sample container is covery with these longer and cooler incubation conditions.not scrupulously clean and contains a low concentration of Whether or not a particular system needs to be monitoredsome microbial nutrient that could promote microbial using high- or low-nutrient media with higher or lower in-growth within the sample container. Because the number of cubation temperatures or shorter or longer incubation timesrecoverable bacteria in a sample can change positively or should be determined during or prior to system validationnegatively over time after sample collection, it is best to test and periodically reassessed as the microbial flora of a newthe samples as soon as possible after being collected. If it is water system gradually establish a steady state relative to itsnot possible to test the sample within about 2 hours of routine maintenance and sanitization procedures. The estab-collection, the sample should be held at refrigerated tem- lishment of a “steady state” can take months or even yearsperatures (2° to 8°) for a maximum of about 12 hours to and can be perturbed by a change in use patterns, amaintain the microbial attributes until analysis. In situations change in routine and preventative maintenance or sanitiza-where even this is not possible (such as when using off-site tion procedures, and frequencies, or any type of system in-contract laboratories), testing of these refrigerated samples trusion, such as for component replacement, removal, orshould be performed within 48 hours after sample collec- addition. The decision to use longer incubation periodstion. In the delayed testing scenario, the recovered micro- should be made after balancing the need for timely infor-bial levels may not be the same as would have been recov- mation and the type of corrective actions required when anered had the testing been performed shortly after sample alert or action level is exceeded with the ability to recovercollection. Therefore, studies should be performed to deter- the microorganisms of interest.mine the existence and acceptability of potential microbial The advantages gained by incubating for longer times,enumeration aberrations caused by protracted testing namely recovery of injured microorganisms, slow growers,delays. or more fastidious microorganisms, should be balanced

against the need to have a timely investigation and to takecorrective action, as well as the ability of these microorgan-

The Classical Culture Approach isms to detrimentally affect products or processes. In nocase, however, should incubation at 30° to 35° be less than

Classical culture approaches for microbial testing of water 48 hours or less than 96 hours at 20° to 25°.include but are not limited to pour plates, spread plates, Normally, the microorganisms that can thrive in extrememembrane filtration, and most probable number (MPN) environments are best cultivated in the laboratory usingtests. These methods are generally easy to perform, are less conditions simulating the extreme environments from whichexpensive, and provide excellent sample processing they were taken. Therefore, thermophilic bacteria might bethroughput. Method sensitivity can be increased via the use able to exist in the extreme environment of hot pharmaceu-of larger sample sizes. This strategy is used in the mem- tical water systems, and if so, could only be recovered andbrane filtration method. Culture approaches are further de- cultivated in the laboratory if similar thermal conditionsfined by the type of medium used in combination with the were provided. Thermophilic aquatic microorganisms do ex-incubation temperature and duration. This combination ist in nature, but they typically derive their energy forshould be selected according to the monitoring needs growth from harnessing the energy from sunlight, from oxi-presented by a specific water system as well as its ability to dation/reduction reactions of elements such as sulfur orrecover the microorganisms of interest: those that could iron, or indirectly from other microorganisms that do derivehave a detrimental effect on the product or process uses as their energy from these processes. Such chemical/nutritionalwell as those that reflect the microbial control status of the conditions do not exist in high purity water systems,system. whether ambient or hot. Therefore, it is generally consid-

There are two basic forms of media available for tradi- ered pointless to search for thermophiles from hot pharma-tional microbiological analysis: “high nutrient” and “low nu- ceutical water systems owing to their inability to growtrient”. High-nutrient media such as plate count agar there.(TGYA) and m-HPC agar (formerly m-SPC agar), are in- The microorganisms that inhabit hot systems tend to betended as general media for the isolation and enumeration found in much cooler locations within these systems, forof heterotrophic or “copiotrophic” bacteria. Low-nutrient example, within use-point heat exchangers or transfer hoses.media such as R2A agar and NWRI agar (HPCA), may be If this occurs, the kinds of microorganisms recovered are




usually of the same types that might be expected from am- Pour Plate Method or Membrane Filtrationbient water systems. Therefore, the mesophilic microbial cul- Drinking Water: Method1

tivation conditions described later in this chapter are usually Sample Volume—1.0 mL minimum2

adequate for their recovery. Growth Medium—Plate Count Agar3

Incubation Time—48 to 72 hours minimumIncubation Temperature—30° to 35°“Instrumental” Approaches

Pour Plate Method or Membrane FiltrationPurified Water: Method1Examples of instrumental approaches include microscopic

Sample Volume—1.0 mL minimum2visual counting techniques (e.g., epifluorescence and immu-Growth Medium—Plate Count Agar3nofluorescence) and similar automated laser scanning ap-Incubation Time—48 to 72 hours minimumproaches and radiometric, impedometric, and biochemicallyIncubation Temperature—30° to 35°based methodologies. These methods all possess a variety of

advantages and disadvantages. Advantages could be their Water for Injection: Membrane Filtration Method1

precision and accuracy or their speed of test result availabil- Sample Volume—100 mL minimum2

ity as compared to the classical cultural approach. In gen- Growth Medium—Plate Count Agar3

eral, instrument approaches often have a shorter lead time Incubation Time—48 to 72 hours minimumfor obtaining results, which could facilitate timely system Incubation Temperature—30°C to 35°Ccontrol. This advantage, however, is often counterbalanced 1 A membrane filter with a rating of 0.45 µm is generally consideredby limited sample processing throughput due to extended preferable even though the cellular width of some of the bacteria insample collection time, costly and/or labor-intensive sample the sample may be narrower than this. The efficiency of the filtrationprocessing, or other instrument and sensitivity limitations. process still allows the retention of a very high percentage of these

Furthermore, instrumental approaches are typically de- smaller cells and is adequate for this application. Filters with smallerstructive, precluding subsequent isolate manipulation for ratings may be used if desired, but for a variety of reasons the abilitycharacterization purposes. Generally, some form of microbial of the retained cells to develop into visible colonies may be compro-isolate characterization, if not full identification, may be a mised, so count accuracy must be verified by a reference approach.required element of water system monitoring. Conse- 2 When colony counts are low to undetectable using the indicatedquently, culturing approaches have traditionally been pre- minimum sample volume, it is generally recognized that a largerferred over instrumental approaches because they offer a sample volume should be tested in order to gain better assurancebalance of desirable test attributes and post-test capabilities. that the resulting colony count is more statistically representative.

The sample volume to consider testing is dependent on the user’sneed to know (which is related to the established alert and actionSuggested Methodologieslevels and the water system’s microbial control capabilities) and thestatistical reliability of the resulting colony count. In order to test aThe following general methods were originally derivedlarger sample volume, it may be necessary to change testing tech-from Standard Methods for the Examination of Water andniques, e.g., changing from a pour plate to a membrane filtrationWastewater, 17th Edition, American Public Health Association,approach. Nevertheless, in a very low to nil count scenario, a maxi-Washington, DC 20005. Even though this publication hasmum sample volume of around 250 to 300 mL is usually consideredundergone several revisions since its first citation in thisa reasonable balance of sample collecting and processing ease andchapter, the methods are still considered appropriate for es-increased statistical reliability. However, when sample volumes largertablishing trends in the number of colony-forming units ob-than about 2 mL are needed, they can only be processed using theserved in the routine microbiological monitoring of pharma-membrane filtration method.ceutical waters. It is recognized, however, that other3 Also known as Standard Methods Agar, Standard Methods Platecombinations of media and incubation time and tempera-Count Agar, or TGYA, this medium contains tryptone (pancreatic di-ture may occasionally or even consistently result in highergest of casein), glucose and yeast extract.numbers of colony-forming units being observed and/or dif-

ferent species being recovered. IDENTIFICATION OF MICROORGANISMSThe extended incubation periods that are usually requiredby some of the alternative methods available offer disadvan- Identifying the isolates recovered from water monitoringtages that may outweigh the advantages of the higher methods may be important in instances where specificcounts that may be obtained. The somewhat higher base- waterborne microorganisms may be detrimental to theline counts that might be observed using alternate cultural products or processes in which the water is used. Microor-conditions would not necessarily have greater utility in de- ganism information such as this may also be useful whentecting an excursion or a trend. In addition, some alternate identifying the source of microbial contamination in a prod-cultural conditions using low-nutrient media tend to lead to uct or process. Often a limited group of microorganisms isthe development of microbial colonies that are much less routinely recovered from a water system. After repeated re-differentiated in colonial appearance, an attribute that mi- covery and characterization, an experienced microbiologistcrobiologists rely on when selecting representative microbial may become proficient at their identification based on onlytypes for further characterization. It is also ironical that the a few recognizable traits such as colonial morphology andnature of some of the slow growers and the extended incu- staining characteristics. This may allow for a reduction in thebation times needed for their development into visible colo- number of identifications to representative colony types, or,nies may also lead to those colonies being largely nonviable, with proper analyst qualification, may even allow testingwhich limits their further characterization and precludes short cuts to be taken for these microbial identifications.their subculture and identification.

Methodologies that can be suggested as generally satis-factory for monitoring pharmaceutical water systems are as ALERT AND ACTION LEVELS ANDfollows. However, it must be noted that these are not refe- SPECIFICATIONSree methods nor are they necessarily optimal for recoveringmicroorganisms from all water systems. The users should Though the use of alert and action levels is most oftendetermine through experimentation with various approaches associated with microbial data, they can be associated withwhich methodologies are best for monitoring their water any attribute. In pharmaceutical water systems, almost everysystems for in-process control and quality control purposes quality attribute, other than microbial quality, can be veryas well as for recovering any contraindicated species theymay have specified.




rapidly determined with near-real time results. These short- process back into its normal operating range. Such remedialdelay data can give immediate system performance feed- actions should also include efforts to understand and elimi-back, serving as ongoing process control indicators. How- nate or at least reduce the incidence of a future occurrence.ever, because some attributes may not continuously be A root cause investigation may be necessary to devise anmonitored or have a long delay in data availability (like mi- effective preventative action strategy. Depending on the na-crobial monitoring data), properly established Alert and Ac- ture of the action level excursion, it may also be necessarytion Levels can serve as an early warning or indication of a to evaluate its impact on the water uses during that time.potentially approaching quality shift occurring between or Impact evaluations may include delineation of affectedat the next periodic monitoring. In a validated water sys- batches and additional or more extensive product testing. Ittem, process controls should yield relatively constant and may also involve experimental product challenges.more than adequate values for these monitored attributes Alert and action levels should be derived from an evalua-such that their Alert and Action Levels are infrequently tion of historic monitoring data called a trend analysis.broached. Other guidelines on approaches that may be used, ranging

As process control indicators, alert and action levels are from “inspectional” to statistical evaluation of the historicaldesigned to allow remedial action to occur that will prevent data have been published. The ultimate goal is to under-a system from deviating completely out of control and pro- stand the normal variability of the data during what is con-ducing water unfit for its intended use. This “intended use” sidered a typical operational period. Then, trigger points orminimum quality is sometimes referred to as a “specifica- levels can be established that will signal when future datation” or “limit”. In the opening paragraphs of this chapter, may be approaching (alert level) or exceeding (action level)rationale was presented for no microbial specifications being the boundaries of that “normal variability”. Such alert andincluded within the body of the bulk water (Purified Water action levels are based on the control capability of the sys-and Water for Injection) monographs. This does not mean tem as it was being maintained and controlled during thatthat the user should not have microbial specifications for historic period of typical control.these waters. To the contrary, in most situations such speci- In new water systems where there is very limited or nofications should be established by the user. The microbial historic data from which to derive data trends, it is commonspecification should reflect the maximum microbial level at to simply establish initial alert and action levels based on awhich the water is still fit for use without compromising the combination of equipment design capabilities but below thequality needs of the process or product where the water is process and product specifications where water is used. It isused. Because water from a given system may have many also common, especially for ambient water systems, to mi-uses, the most stringent of these uses should be used to crobiologically “mature” over the first year of use. By theestablish this specification. end of this period, a relatively steady state microbial popula-

Where appropriate, a microbial specification could be tion (microorganism types and levels) will have been al-qualitative as well as quantitative. In other words, the num- lowed or promoted to develop as a result of the collectiveber of total microorganisms may be as important as the effects of routine system maintenance and operation, includ-number of a specific microorganism or even the absence of ing the frequency of unit operation rebeddings, backwash-a specific microorganism. Microorganisms that are known to ings, regenerations, and sanitizations. This microbial popula-be problematic could include opportunistic or overt patho- tion will typically be higher than was seen when the watergens, nonpathogenic indicators of potentially undetected system was new, so it should be expected that the datapathogens, or microorganisms known to compromise a pro- trends (and the resulting alert and action levels) will increasecess or product, such as by being resistant to a preservative over this “maturation” period and eventually level off.or able to proliferate in or degrade a product. These micro- A water system should be designed so that performance-organisms comprise an often ill-defined group referred to as based alert and action levels are well below water specifica-“objectionable microorganisms”. Because objectionable is a tions. With poorly designed or maintained water systems,term relative to the water’s use, the list of microorganisms the system owner may find that initial new system microbialin such a group should be tailored to those species with the levels were acceptable for the water uses and specifications,potential to be present and problematic. Their negative im- but the mature levels are not. This is a serious situation,pact is most often demonstrated when they are present in which if not correctable with more frequent system mainte-high numbers, but depending on the species, an allowable nance and sanitization, may require expensive water systemlevel may exist, below which they may not be considered renovation or even replacement. Therefore, it cannot beobjectionable. overemphasized that water systems should be designed for

As stated above, alert and action levels for a given process ease of microbial control, so that when monitored againstcontrol attribute are used to help maintain system control alert and action levels, and maintained accordingly, theand avoid exceeding the pass/fail specification for that attri- water continuously meets all applicable specifications.bute. Alert and action levels may be both quantitative and An action level should not be established at a level equiv-qualitative. They may involve levels of total microbial counts alent to the specification. This leaves no room for remedialor recoveries of specific microorganisms. Alert levels are system maintenance that could avoid a specification excur-events or levels that, when they occur or are exceeded, indi- sion. Exceeding a specification is a far more serious eventcate that a process may have drifted from its normal operat- than an action level excursion. A specification excursion maying condition. Alert level excursions constitute a warning trigger an extensive finished product impact investigation,and do not necessarily require a corrective action. However, substantial remedial actions within the water system thatalert level excursions usually lead to the alerting of person- may include a complete shutdown, and possibly even prod-nel involved in water system operation as well as QA. Alert uct rejection.level excursions may also lead to additional monitoring with Another scenario to be avoided is the establishment of anmore intense scrutiny of resulting and neighboring data as arbitrarily high and usually nonperformance based actionwell as other process indicators. Action levels are events or level. Such unrealistic action levels deprive users of mean-higher levels that, when they occur or are exceeded, indi- ingful indicator values that could trigger remedial systemcate that a process is probably drifting from its normal oper- maintenance. Unrealistically high action levels allow systemsating range. Examples of kinds of action level “events” in- to grow well out of control before action is taken, whenclude exceeding alert levels repeatedly; or in multiple their intent should be to catch a system imbalance before itsimultaneous locations, a single occurrence of exceeding a goes wildly out of control.higher microbial level; or the individual or repeated recovery Because alert and action levels should be based on actualof specific objectionable microorganisms. Exceeding an ac- system performance, and the system performance data aretion level should lead to immediate notification of both QA generated by a given test method, it follows that those alertand personnel involved in water system operations so that and action levels should be valid only for test results gener-corrective actions can immediately be taken to bring the ated by the same test method. It is invalid to apply alert



USP 35 General Information / ⟨1235⟩ Vaccines for Human Use—General Considerations 907

and action level criteria to test results generated by a differ- Examples of types of licensed vaccines appear in Appendixent test method. The two test methods may not equiva- 1. A current list of vaccines licensed in the United States islently recover microorganisms from the same water samples. posted at www.fda.gov/cber/.Similarly invalid is the use of trend data to derive alert and Vaccines can be of various types, depending on their de-action levels for one water system, but applying those alert sign and processes involved in their manufacture. Vaccinesand action levels to a different water system. Alert and ac- for human use may contain whole killed or attenuated or-tion levels are water system and test method specific. ganisms (e.g., bacteria or viruses) or contain antigens de-

Nevertheless, there are certain maximum microbial levels rived from portions of a pathogen, either by partitioningabove which action levels should never be established. and purification or derived using recombinant technologyWater systems with these levels should unarguably be con- (Table 1). Some polysaccharide vaccines are conjugated to asidered out of control. Using the microbial enumeration carrier in order to enhance their immune response.methodologies suggested above, generally considered maxi-mum action levels are 100 cfu per mL for Purified Water and Table 1. Bacterial and Viral Vaccines10 cfu per 100 mL for Water for Injection. However, if a

Live attenuated whole cell or virusagiven water system controls microorganisms much moretightly than these levels, appropriate alert and action levels Inactivated/killedb

should be established from these tighter control levels so Whole cell or virusc

that they can truly indicate when water systems may be Recombinant proteinsd

starting to trend out of control. These in-process microbialSubunitecontrol parameters should be established well below the

Polysaccharidesuser-defined microbial specifications that delineate the wa-Proteinster’s fitness for use.

Special consideration is needed for establishing maximum Modified toxinsmicrobial action levels for Drinking Water because the water a Live attenuated bacterial or viral vaccines are weakened (attenuated)is often delivered to the facility in a condition over which forms of a pathogen. They contain antigens that are similar to disease-the user has little control. High microbial levels in Drinking causing microbes. They may be derived from the pathogen itself, orWater may be indicative of a municipal water system upset, from a different organism that contains antigens that cross-react withbroken water main, or inadequate disinfection, and there- the virulent microbe (e.g., vaccinia and variola).fore, potential contamination with objectionable microor- b Inactivated bacterial and viral vaccines are produced by growing cellsganisms. Using the suggested microbial enumeration meth- of disease-causing bacteria or viruses in cell substrates and subsequentlyodology, a reasonable maximum action level for Drinking inactivating them to prevent replication in the recipient.Water is 500 cfu per mL. Considering the potential concern

c Inactivated/killed whole-cell or virus vaccines consist of the entire mi-for objectionable microorganisms raised by such high micro-croorganisms after they have been inactivated. These preparations maybial levels in the feedwater, informing the municipality ofor may not be partially or completely purified.the problem so they may begin corrective actions should bed Recombinant protein viral and bacterial vaccines are derived from hostan immediate first step. In-house remedial actions may orcells that have been transformed with expression vectors that carrymay not also be needed, but could include performing ad-genes that encode antigenic material from infectious agents. The expres-ditional coliform testing on the incoming water and pre-sion cells are grown in bioreactors to produce the recombinant antigen-treating the water with either additional chlorination or UVic material.light irradiation or filtration or a combination of approaches.e Subunit vaccines are extracts from inactivated/killed viruses or bacte-ria. Subunit-type vaccines generally undergo some degree of purifica-tion.

In addition to antigen(s), vaccines may contain severalother components, such as adjuvants that enhance the im-mune response to the vaccine antigen, preservatives to pre-⟨1235⟩ VACCINES FOR HUMAN vent bacterial or fungal contamination of multiple-dose vials,or other excipients needed for pharmaceutical manufactur-USE—GENERAL ing or vaccine stabilization. Residual components from themanufacturing process also may be present in vaccine prep-CONSIDERATIONS arations. Examples of these categories are listed in Table 2.

Table 2. Vaccine Components

AntigensINTRODUCTION Whole organisms

Components/subunitsVaccines have been used for centuries to immunize indi- Recombinant proteinsviduals against pathogenic organisms with the goal of

Adjuvantspreventing the associated disease. Vaccines are biologicalAluminum saltsproducts that contain antigens capable of inducing a spe-

Antimicrobial preservativescific and active acquired immune response in the body. An-tigens present in vaccines are processed by specialized cells Thimerosalin the body’s immune system, resulting in the development 2-Phenoxyethanolof blood proteins known as antibodies (i.e., humoral immu- Benzethonium chloridenity) or specialized lymphocytes (i.e., cell-mediated immu-

Phenolnity) or both. Therefore immune responses may be antibodyStabilizersmediated, cell mediated, or both. Thus, antigens are critical

Saltsfor vaccine function and generally consist of a portion of thepathogenic organism, or an attenuated form of the whole Amino acidsmicroorganism. In the case of DNA-based vaccines (cur- Sugarsrently under development), the vaccine would contain nu- Proteinscleotide sequences (genetic material) that encode microbial

Otherantigens.



1231 Water for Pharmaceutical Purposes Usp35

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