The following waters are typically produced in large volume by a multiple-unit operation water system and distributed by a piping system for use at the same site. These particular pharmaceutical waters must meet the quality attributes as specified in the related monographs.

The following monographed waters are packaged forms of either Purified Water or Water for Injection that have been sterilized to preserve their microbiological properties. These waters may have specific intended uses as indicated by their names and may also have restrictions on packaging configurations related to those uses. In general, these packaged waters may be used in lieu of the bulk form of water from which they were derived. However, the user should take into consideration that the packaging and sterilization processes used for the articles may leach materials from the packaging material into the water over its shelf life, rendering it less pure than the original water placed into the package. The chemical attributes of these waters are still defined primarily by the wet chemistry methods and specifications similar to those formerly used for the bulk pharmaceutical waters prior to their replacement with water conductivity and total organic carbon (TOC). It is the user's responsibility to ensure fitness for use of this article when used in manufacturing, clinical, or analytical applications where the purer bulk form of the water is indicated.

In addition to the bulk monographed waters described above, nonmonographed waters can also be used in pharmaceutical processing steps such as cleaning, synthetic steps or as a starting material for further purification. The following is a description of several of these nonmonographed waters as cited in various locations within this compendia.

Nonmonographed Analytical Waters

Distilled Water— This water is produced by vaporizing liquid water and condensing it in a purer state. It is used primarily as a solvent for reagent preparation, but it is also specified in the execution of other aspects of tests, such as for rinsing an analyte, transferring a test material as a slurry, as a calibration standard or analytical blank, and for test apparatus cleaning. It is also cited as the starting water to be used for making High Purity Water. Because none of the cited uses of this water imply a need for a particular purity attribute that can only be derived by distillation, water meeting the requirements for Purified Water derived by other means of purification could be equally suitable where Distilled Water is specified.

Freshly Distilled Water— Also called “recently distilled water”, it is produced in a similar fashion to Distilled Water and should be used shortly after its generation. This implies the need to avoid endotoxin contamination as well as any other adventitious forms of contamination from the air or containers that could arise with prolonged storage. It is used for preparing solutions for subcutaneous test animal injections as well as for a reagent solvent in tests for which there appears to be no particularly high water purity needed that could be ascribable to being “freshly distilled”. In the “test-animal” use, the term “freshly distilled” and its testing use imply a chemical, endotoxin, and microbiological purity that could be equally satisfied by Water for Injection (though no reference is made to these chemical, endotoxin, or microbial attributes or specific protection from recontamination). For nonanimal uses, water meeting the requirements for Purified Water derived by other means of purification and/or storage periods could be equally suitable where “recently distilled water” or Freshly Distilled Water is specified.

Deionized Water— This water is produced by an ion-exchange process in which the contaminating ions are replaced with either H+ or OH– ions. Similarly to Distilled Water, Deionized Water is used primarily as a solvent for reagent preparation, but it is also specified in the execution of other aspects of tests, such as for transferring an analyte within a test procedure, as a calibration standard or analytical blank, and for test apparatus cleaning. Also, none of the cited uses of this water imply any needed purity attribute that can only be achieved by deionization. Therefore, water meeting the requirements for Purified Water that is derived by other means of purification could be equally suitable where Deionized Water is specified.

Freshly Deionized Water— This water is prepared in a similar fashion to Deionized Water, though as the name suggests, it is to be used shortly after its production. This implies the need to avoid any adventitious contamination that could occur upon storage. This water is indicated for use as a reagent solvent as well as for cleaning. Due to the nature of the testing, Purified Water could be a reasonable alternative for these applications.

Deionized Distilled Water— This water is produced by deionizing (see Deionized Water ) Distilled Water. This water is used as a reagent in a liquid chromatography test that requires a high purity. Because of the importance of this high purity, water that barely meets the requirements for Purified Water may not be acceptable. High Purity Water (see below) could be a reasonable alternative for this water.

Filtered Distilled or Deionized Water— This water is essentially Purified Water produced by distillation or deionization that has been filtered through a 1.2-µm rated membrane. This water is used in particulate matter testing where the presence of particles in the water could bias the test results (see Particulate Matter in Injections 788). Because the chemical water purity needed for this test could also be afforded by water purification processes other than distillation or deionization, filtered water meeting the requirements for Purified Water, but produced by means other than distillation or deionization could be equally suitable.

Filtered Water— This water is Purified Water that has been filtered to remove particles that could interfere with the analysis where the water is used. Where used for preparing samples for particulate matter testing (see Particulate Matter in Injections 788), though unspecified in monographs, water filtration should be through a 1.2-µm filter to be consistent with the general test chapter. Where used as a chromatography reagent, monograph-specified filter ratings range from 0.5 µm to unspecified.

High Purity Water— The preparation of this water is defined in Containers 661. It is water that is prepared by deionizing previously distilled water, and then filtering it through a 0.45-µm rated membrane. This water must have an in-line conductivity of not greater than 0.15 µS/cm (6.67 Megohm-cm) at 25. For the sake of purity comparison, the analogous Stage 1 and 2 conductivity requirements for Purified Water at the same temperature are 1.3 µS/cm and 2.1 µS/cm, respectively. The preparation specified in Containers 661 uses materials that are highly efficient deionizers and that do not contribute copper ions or organics to the water, assuring a very high quality water. If the water of this purity contacts the atmosphere even briefly as it is being used or drawn from its purification system, its conductivity will immediately degrade, by as much as about 1.0 µS/cm, as atmospheric carbon dioxide dissolves in the water and equilibrates to bicarbonate ions. Therefore, if the analytical use requires that water purity remains as high as possible, its use should be protected from atmospheric exposure. This water is used as a reagent, as a solvent for reagent preparation, and for test apparatus cleaning where less pure waters would not perform acceptably. However, if a user's routinely available purified water is filtered and meets or exceeds the conductivity specifications of High Purity Water, it could be used in lieu of High Purity Water.

Ammonia-Free Water— Functionally, this water must have a negligible ammonia concentration to avoid interference in tests sensitive to ammonia. It has been equated with High Purity Water that has a significantly tighter Stage 1 conductivity specification than Purified Water because of the latter's allowance for a minimal level of ammonium among other ions. However, if the user's Purified Water were filtered and met or exceeded the conductivity specifications of High Purity Water, it would contain negligible ammonia or other ions and could be used in lieu of High Purity Water.

Carbon Dioxide-Free Water— The introductory portion of the Reagents, Indicators, and Solutions section defines this water as Purified Water that has been vigorously boiled for at least 5 minutes, then cooled and protected from absorption of atmospheric carbon dioxide. Because the absorption of carbon dioxide tends to drive down the water pH, most of the uses of Carbon Dioxide-Free Water are either associated as a solvent in pH-related or pH- sensitive determinations or as a solvent in carbonate-sensitive reagents or determinations. Another use of this water is for certain optical rotation and color and clarity of solution tests. Though it is possible that this water is indicated for these tests simply because of its purity, it is also possible that the pH effects of carbon dioxide containing water could interfere with the results of these tests. A third plausible reason that this water is indicated is that outgassing air bubbles might interfere with these photometric-type tests. The boiled water preparation approach will also greatly reduced the concentrations of many other dissolved gases along with carbon dioxide. Therefore, in some of the applications for Carbon Dioxide-Free Water, it could be the inadvertent deaeration effect that actually renders this water suitable. In addition to boiling, deionization is perhaps an even more efficient process for removing dissolved carbon dioxide (by drawing the dissolved gas equilibrium toward the ionized state with subsequent removal by the ion-exchange resins). If the starting Purified Water is prepared by an efficient deionization process and protected after deionization from exposure to atmospheric air, water that is carbon dioxide-free can be effectively made without the application of heat. However this deionization process does not deaerate the water, so if Purified Water prepared by deionization is considered as a substitute water in a test requiring Carbon Dioxide-Free Water, the user must verify that it is not actually water akin to Deaerated Water (discussed below) that is needed for the test. As indicated in the High Purity Water, even brief contact with the atmosphere can allow small amounts of carbon dioxide to dissolve, ionize, and significantly degrade the conductivity and lower the pH. If the analytical use requires the water to remain as pH-neutral and as carbon dioxide-free as possible, even the analysis should be protected from atmospheric exposure. However, in most applications, atmospheric exposure during testing does not significantly affect its suitability in the test.

Ammonia- and Carbon Dioxide-Free Water— As implied by the name, this water should be prepared by approaches compatible with those mentioned for both Ammonia-Free Water and Carbon Dioxide-Free Water. Because the carbon dioxide-free attribute requires post-production protection from the atmosphere, it is appropriate to first render the water ammonia-free using the High Purity Water process followed by the boiling and carbon dioxide-protected cooling process. The High Purity Water deionization process for creating Ammonia-Free Water will also remove the ions generated from dissolved carbon dioxide and ultimately, by forced equilibration to the ionized state, all the dissolved carbon dioxide. Therefore, depending on its use, an acceptable procedure for making Ammonia- and Carbon Dioxide-Free Water could be to transfer and collect High Purity Water in a carbon dioxide intrusion-protected container.

Deaerated Water— This water is Purified Water that has been treated to reduce the content of dissolved air by “suitable means”. In the Reagents section, approaches for boiling, cooling (similar to Carbon Dioxide-Free Water but without the atmospheric carbon dioxide protection), and sonication are given as applicable for test uses other than dissolution and drug release testing. Though Deaerated Water is not mentioned by name in Dissolution 711, suggested methods for deaerating dissolution media (which may be water) include warming to 41, vacuum filtering through a 0.45-µm rated membrane, and vigorously stirring the filtrate while maintaining the vacuum. This chapter specifically indicates that other validated approaches may be used. In other monographs that also do not mention Deaerated Water by name, degassing of water and other reagents is accomplished by sparging with helium. Deaerated Water is used in both dissolution testing as well as liquid chromatography applications where outgassing could either interfere with the analysis itself or cause erroneous results due to inaccurate volumetric withdrawals. Applications where ambient temperature water is used for reagent preparation, but the tests are performed at elevated temperatures, are candidates for outgassing effects. If outgassing could interfere with test performance, including, chromatographic flow, colorimetric, or photometric measurements, or volumetric accuracy, then Deaerated Water should probably be used, whether called for in the analysis or not. The above deaeration approaches might not render the water “gas-free”. At best, they reduce the dissolved gas concentrations so that outgassing caused by temperature changes is not likely.

Recently Boiled Water— This water may include recently or freshly boiled water (with or without mention of cooling in the title), but cooling prior to use is clearly intended. Occasionally it is necessary to use when hot. Recently Boiled Water is specified because it is used in a pH-related test or carbonate-sensitive reagent, in an oxygen-sensitive test or reagent, or in a test where outgassing could interfere with the analysis, such as specific gravity or an appearance test.

The purpose of prefiltration—also referred to as initial, coarse, or depth filtration—is to remove solid contaminants down to a size of 7 to 10 µm from the incoming source water supply and protect downstream system components from particulates that can inhibit equipment performance and shorten their effective life. This coarse filtration technology utilizes primarily sieving effects for particle capture and a depth of filtration medium that has a high “dirt load” capacity. Such filtration units are available in a wide range of designs and for various applications. Removal efficiencies and capacities differ significantly, from granular bed filters such as multimedia or sand for larger water systems, to depth cartridges for smaller water systems. Unit and system configurations vary widely in type of filtering media and location in the process. Granular or cartridge prefilters are often situated at or near the head of the water pretreatment system prior to unit operations designed to remove the source water disinfectants. This location, however, does not preclude the need for periodic microbial control because biofilm can still proliferate, although at a slower rate in the presence of source water disinfectants. Design and operational issues that may impact performance of depth filters include channeling of the filtering media, blockage from silt, microbial growth, and filtering-media loss during improper backwashing. Control measures involve pressure and flow monitoring during use and backwashing, sanitizing, and replacing filtering media. An important design concern is sizing of the filter to prevent channeling or media loss resulting from inappropriate water flow rates as well as proper sizing to minimize excessively frequent or infrequent backwashing or cartridge filter replacement.

Granular activated carbon beds adsorb low molecular weight organic material and oxidizing additives, such as chlorine and chloramine compounds, removing them from the water. They are used to achieve certain quality attributes and to protect against reaction with downstream stainless steel surfaces, resins, and membranes. The chief operating concerns regarding activated carbon beds include the propensity to support bacteria growth, the potential for hydraulic channeling, the organic adsorption capacity, appropriate water flow rates and contact time, the inability to be regenerated in situ, and the shedding of bacteria, endotoxins, organic chemicals, and fine carbon particles. Control measures may involve monitoring water flow rates and differential pressures, sanitizing with hot water or steam, backwashing, testing for adsorption capacity, and frequent replacement of the carbon bed. If the activated carbon bed is intended for organic reduction, it may also be appropriate to monitor influent and effluent TOC. It is important to note that the use of steam for carbon bed sanitization is often incompletely effective due to steam channeling rather than even permeation through the bed. This phenomenon can usually be avoided by using hot water sanitization. It is also important to note that microbial biofilm development on the surface of the granular carbon particles (as well as on other particles such as found in deionizer beds and even multimedia beds) can cause adjacent bed granules to “stick” together. When large masses of granules are agglomerated in this fashion, normal backwashing and bed fluidization flow parameters may not be sufficient to disperse them, leading to ineffective removal of trapped debris, loose biofilm, and penetration of microbial controlling conditions (as well as regenerant chemicals as in the case of agglomerated deionizer resins). Alternative technologies to activated carbon beds can be used in order to avoid their microbial problems, such as disinfectant-neutralizing chemical additives and regenerable organic scavenging devices. However, these alternatives do not function by the same mechanisms as activated carbon, may not be as effective at removing disinfectants and some organics, and have a different set of operating concerns and control measures that may be nearly as troublesome as activated carbon beds.

Chemical additives are used in water systems (a) to control microorganisms by use of sanitants such as chlorine compounds and ozone, (b) to enhance the removal of suspended solids by use of flocculating agents, (c) to remove chlorine compounds, (d) to avoid scaling on reverse osmosis membranes, and (e) to adjust pH for more effective removal of carbonate and ammonia compounds by reverse osmosis. These additives do not constitute “added substances” as long as they are either removed by subsequent processing steps or are otherwise absent from the finished water. Control of additives to ensure a continuously effective concentration and subsequent monitoring to ensure their removal should be designed into the system and included in the monitoring program.

Organic scavenging devices use macroreticular weakly basic anion-exchange resins capable of removing organic material and endotoxins from the water. They can be regenerated with appropriate biocidal caustic brine solutions. Operating concerns are associated with organic scavenging capacity, particulate, chemical and microbiological fouling of the reactive resin surface, flow rate, regeneration frequency, and shedding of resin fragments. Control measures include TOC testing of influent and effluent, backwashing, monitoring hydraulic performance, and using downstream filters to remove resin fines.

Water softeners may be located either upstream or downstream of disinfectant removal units. They utilize sodium-based cation-exchange resins to remove water-hardness ions, such as calcium and magnesium, that could foul or interfere with the performance of downstream processing equipment such as reverse osmosis membranes, deionization devices, and distillation units. Water softeners can also be used to remove other lower affinity cations, such as the ammonium ion, that may be released from chloramine disinfectants commonly used in drinking water and which might otherwise carryover through other downstream unit operations. If ammonium removal is one of its purposes, the softener must be located downstream of the disinfectant removal operation, which itself may liberate ammonium from neutralized chloramine disinfectants. Water softener resin beds are regenerated with concentrated sodium chloride solution (brine). Concerns include microorganism proliferation, channeling caused by biofilm agglomeration of resin particles, appropriate water flow rates and contact time, ion-exchange capacity, organic and particulate resin fouling, organic leaching from new resins, fracture of the resin beads, resin degradation by excessively chlorinated water, and contamination from the brine solution used for regeneration. Control measures involve recirculation of water during periods of low water use, periodic sanitization of the resin and brine system, use of microbial control devices (e.g., UV light and chlorine), locating the unit upstream of the disinfectant removal step (if used only for softening), appropriate regeneration frequency, effluent chemical monitoring (e.g., hardness ions and possibly ammonium), and downstream filtration to remove resin fines. If a softener is used for ammonium removal from chloramine-containing source water, then capacity, contact time, resin surface fouling, pH, and regeneration frequency are very important.

Deionization (DI), and continuous electrodeionization (CEDI) are effective methods of improving the chemical quality attributes of water by removing cations and anions. DI systems have charged resins that require periodic regeneration with an acid and base. Typically, cationic resins are regenerated with either hydrochloric or sulfuric acid, which replace the captured positive ions with hydrogen ions. Anionic resins are regenerated with sodium or potassium hydroxide, which replace captured negative ions with hydroxide ions. Because free endotoxin is negatively charged, there is some removal of endotoxin achieved by the anionic resin. Both regenerant chemicals are biocidal and offer a measure of microbial control. The system can be designed so that the cation and anion resins are in separate or “twin” beds or they can be mixed together to form a mixed bed. Twin beds are easily regenerated but deionize water less efficiently than mixed beds, which have a considerably more complex regeneration process. Rechargeable resin canisters can also be used for this purpose.

The CEDI system uses a combination of mixed resin, selectively permeable membranes, and an electric charge, providing continuous flow (product and waste concentrate) and continuous regeneration. Water enters both the resin section and the waste (concentrate) section. As it passes through the resin, it is deionized to become product water. The resin acts as a conductor enabling the electrical potential to drive the captured cations and anions through the resin and appropriate membranes for concentration and removal in the waste water stream. The electrical potential also separates the water in the resin (product) section into hydrogen and hydroxide ions. This permits continuous regeneration of the resin without the need for regenerant additives. However, unlike conventional deionization, CEDI units must start with water that is already partially purified because they generally cannot produce Purified Water quality when starting with the heavier ion load of unpurified source water.

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

Reverse osmosis (RO) units employ semipermeable membranes. The “pores” of RO membranes are actually intersegmental spaces among the polymer molecules. They are big enough for permeation of water molecules, but too small to permit passage of hydrated chemical ions. However, many factors including pH, temperature, and differential pressure across the membrane affect the selectivity of this permeation. With the proper controls, RO membranes can achieve chemical, microbial, and endotoxin quality improvement. The process streams consist of supply water, product water (permeate), and wastewater (reject). Depending on source water, pretreatment and system configuration variations and chemical additives may be necessary to achieve desired performance and reliability.

A major factor affecting RO performance is the permeate recovery rate, that is, the amount of the water passing through the membrane compared to the amount rejected. This is influenced by the several factors, but most significantly by the pump pressure. Recoveries of 75% are typical, and can accomplish a 1 to 2 log purification of most impurities. For most feed waters, this is usually not enough to meet Purified Water conductivity specifications. A second pass of this permeate water through another RO stage usually achieves the necessary permeate purity if other factors such as pH and temperature have been appropriately adjusted and the ammonia from chloraminated source water has been previously removed. Increasing recoveries with higher pressures in order to reduce the volume of reject water will lead to reduced permeate purity. If increased pressures are needed over time to achieve the same permeate flow, this is an indication of partial membrane blockage that needs to be corrected before it becomes irreversibly fouled, and expensive membrane replacement is the only option.

Other concerns associated with the design and operation of RO units include membrane materials that are extremely sensitive to sanitizing agents and to particulate, chemical, and microbial membrane fouling; membrane and seal integrity; the passage of dissolved gases, such as carbon dioxide and ammonia; and the volume of wastewater, particularly where water discharge is tightly regulated by local authorities. Failure of membrane or seal integrity will result in product water contamination. Methods of control involve suitable pretreatment of the influent water stream, appropriate membrane material selection, integrity challenges, membrane design and heat tolerance, periodic sanitization, and monitoring of differential pressures, conductivity, microbial levels, and TOC.

The development of RO units that can tolerate sanitizing water temperatures as well as operate efficiently and continuously at elevated temperatures has added greatly to their microbial control and to the avoidance of biofouling. RO units can be used alone or in combination with DI and CEDI units as well as ultrafiltration for operational and quality enhancements.

Ultrafiltration is a technology most often employed in pharmaceutical water systems for removing endotoxins from a water stream. It can also use semipermeable membranes, but unlike RO, these typically use polysulfone membranes whose intersegmental “pores” have been purposefully exaggerated during their manufacture by preventing the polymer molecules from reaching their smaller equilibrium proximities to each other. Depending on the level of equilibrium control during their fabrication, membranes with differing molecular weight “cutoffs” can be created such that molecules with molecular weights above these cutoffs ratings are rejected and cannot penetrate the filtration matrix.

Ceramic ultrafilters are another molecular sieving technology. Ceramic ultrafilters are self supporting and extremely durable, backwashable, chemically cleanable, and steam sterilizable. However, they may require higher operating pressures than membrane type ultrafilters.

All ultrafiltration devices work primarily by a molecular sieving principle. Ultrafilters with molecular weight cutoff ratings in the range of 10,000 to 20,000 Da are typically used in water systems for removing endotoxins. This technology may be appropriate as an intermediate or final purification step. Similar to RO, successful performance is dependent upon pretreatment of the water by upstream unit operations.

Issues of concern for ultrafilters include compatibility of membrane material with heat and sanitizing agents, membrane integrity, fouling by particles and microorganisms, and seal integrity. Control measures involve filtration medium selection, sanitization, flow design (dead end vs. tangential), integrity challenges, regular cartridge changes, elevated feed water temperature, and monitoring TOC and differential pressure. Additional flexibility in operation is possible based on the way ultrafiltration units are arranged such as in a parallel or series configurations. Care should be taken to avoid stagnant water conditions that could promote microorganism growth in back-up or standby units.

Charge-modified filters are usually microbially retentive filters that are treated during their manufacture to have a positive charge on their surfaces. Microbial retentive filtration will be described in a subsequent section, but the significant feature of these membranes is their electrostatic surface charge. Such charged filters can reduce endotoxin levels in the fluids passing through them by their adsorption (owing to endotoxin's negative charge) onto the membrane surfaces. Though ultrafilters are more often employed as a unit operation for endotoxin removal in water systems, charge-modified filters may also have a place in endotoxin removal particularly where available upstream pressures are not sufficient for ultrafiltration and for a single, relatively short term use. Charge-modified filters may be difficult to validate for long-term or large-volume endotoxin retention. Even though their purified standard endotoxin retention can be well characterized, their retention capacity for “natural” endotoxins is difficult to gauge. Nevertheless, utility could be demonstrated and validated as short-term, single-use filters at points of use in water systems that are not designed for endotoxin control or where only an endotoxin “polishing” (removal of only slight or occasional endotoxin levels) is needed. Control and validation concerns include volume and duration of use, flow rate, water conductivity and purity, and constancy and concentration of endotoxin levels being removed. All of these factors may have to be evaluated and challenged prior to using this approach, making this a difficult-to-validate application. Even so, there may still be a possible need for additional backup endotoxin testing both upstream and downstream of the filter.

Microbial-retentive membrane filters have experienced an evolution of understanding in the past decade that has caused previously held theoretical retention mechanisms to be reconsidered. These filters have a larger effective “pore size” than ultrafilters and are intended to prevent the passage of microorganisms and similarly sized particles without unduly restricting flow. This type of filtration is widely employed within water systems for filtering the bacteria out of both water and compressed gases as well as for vent filters on tanks and stills and other unit operations. However, the properties of the water system microorganisms seem to challenge a filter's microbial retention from water with phenomena absent from other aseptic filtration applications, such as filter sterilizing of pharmaceutical formulations prior to packaging. In the latter application, sterilizing grade filters are generally considered to have an assigned rating of 0.2 or 0.22 µm. This rather arbitrary rating is associated with filters that have the ability to retain a high level challenge of a specially prepared inoculum of Brevundimonas (formerly Pseudomonas) diminuta. This is a small microorganism originally isolated decades ago from a product that had been “filter sterilized” using a 0.45-µm rated filter. Further study revealed that a percentage of cells of this microorganism could reproducibly penetrate the 0.45-µm sterilizing filters. Through historic correlation of B. diminuta retaining tighter filters, thought to be twice as good as 0.45-µm filter, assigned ratings of 0.2 or 0.22 µm with their successful use in product solution filter sterilization, both this filter rating and the associated high level B. diminuta challenge have become the current benchmarks for sterilizing filtration. New evidence now suggests that for microbial-retentive filters used for pharmaceutical water, B. diminuta may not be the best model microorganism.

An archaic understanding of microbial retentive filtration would lead one to equate a filter's rating with the false impression of a simple sieve or screen that absolutely retains particles sized at or above the filter's rating. A current understanding of the mechanisms involved in microbial retention and the variables that can affect those mechanisms has yielded a far more complex interaction of phenomena than previously understood. A combination of simple sieve retention and surface adsorption are now known to contribute to microbial retention.

The following all interact to create some unusual and surprising retention phenomena for water system microorganisms: the variability in the range and average pore sizes created by the various membrane fabrication processes, the variability of the surface chemistry and three-dimensional structure related to the different polymers used in these filter matrices, and the size and surface properties of the microorganism intended to be retained by the filters. B. diminuta may not the best challenge microorganisms for demonstrating bacterial retention for 0.2- to 0.22-µm rated filters for use in water systems because it appears to be more easily retained by these filters than some water system flora. The well-documented appearance of water system microorganisms on the downstream sides of some 0.2- to 0.22-µm rated filters after a relatively short period of use seems to support that some penetration phenomena are at work. Unknown for certain is if this downstream appearance is caused by a “blow-through” or some other pass-through phenomenon as a result of tiny cells or less cell “stickiness”, or by a “growth through” phenomenon as a result of cells hypothetically replicating their way through the pores to the downstream side. Whatever is the penetration mechanism, 0.2- to 0.22-µm rated membranes may not be the best choice for some water system uses.

Microbial retention success in water systems has been reported with the use of some manufacturers' filters arbitrarily rated as 0.1 µm. There is general agreement that for a given manufacturer, their 0.1-µm rated filters are tighter than their 0.2- to 0.22-µm rated filters. However, comparably rated filters from different manufacturers in water filtration applications may not perform equivalently owing to the different filter fabrication processes and the nonstandardized microbial retention challenge processes currently used for defining the 0.1-µm filter rating. It should be noted that use of 0.1-µm rated membranes generally results in a sacrifice in flow rate compared to 0.2- to 0.22-µm membranes, so whatever membranes are chosen for a water system application, the user must verify that the membranes are suitable for their intended application, use period, and use process, including flow rate.

For microbial retentive gas filtrations, the same sieving and adsorptive retention phenomena are at work as in liquid filtration, but the adsorptive phenomenon is enhanced by additional electrostatic interactions between particles and filter matrix. These electrostatic interactions are so strong that particle retention for a given filter rating is significantly more efficient in gas filtration than in water or product solution filtrations. These additional adsorptive interactions render filters rated at 0.2 to 0.22 µm unquestionably suitable for microbial retentive gas filtrations. When microbially retentive filters are used in these applications, the membrane surface is typically hydrophobic (non-wettable by water). A significant area of concern for gas filtration is blockage of tank vents by condensed water vapor, which can cause mechanical damage to the tank. Control measures include electrical or steam tracing and a self-draining orientation of vent filter housings to prevent accumulation of vapor condensate. However, a continuously high filter temperature will take an oxidative toll on polypropylene components of the filter, so sterilization of the unit prior to initial use, and periodically thereafter, as well as regular visual inspections, integrity tests, and changes are recommended control methods.

In water applications, microbial retentive filters may be used downstream of unit operations that tend to release microorganisms or upstream of unit operations that are sensitive to microorganisms. Microbial retentive filters may also be used to filter water feeding the distribution system. It should be noted that regulatory authorities allow the use of microbial retentive filters within distribution systems or even at use points if they have been properly validated and are appropriately maintained. A point-of-use filter should only be intended to “polish” the microbial quality of an otherwise well-maintained system and not to serve as the primary microbial control device. The efficacy of system microbial control measures can only be assessed by sampling the water upstream of the filters. As an added measure of protection, in-line UV lamps, appropriately sized for the flow rate (see Sanitization), may be used just upstream of microbial retentive filters to inactivate microorganisms prior to their capture by the filter. This tandem approach tends to greatly delay potential microbial penetration phenomena and can substantially extend filter service life.

The use of low-pressure UV lights that emit a 254-nm wavelength for microbial control is discussed under Sanitization, but the application of UV light in chemical purification is also emerging. This 254-nm wavelength is also useful in the destruction of ozone. With intense emissions at wavelengths around 185 nm (as well as at 254 nm), medium pressure UV lights have demonstrated utility in the destruction of the chlorine containing disinfectants used in source water as well as for interim stages of water pretreatment. High intensities of this wavelength alone or in combination with other oxidizing sanitants, such as hydrogen peroxide, have been used to lower TOC levels in recirculating distribution systems. The organics are typically converted to carbon dioxide, which equilibrates to bicarbonate, and incompletely oxidized carboxylic acids, both of which can easily be removed by polishing ion-exchange resins. Areas of concern include adequate UV intensity and residence time, gradual loss of UV emissivity with bulb age, gradual formation of UV-absorbing film at the water contact surface, incomplete photodegradation during unforeseen source water hyperchlorination, release of ammonia from chloramine photodegradation, unapparent UV bulb failure, and conductivity degradation in distribution systems using 185-nm UV lights. Control measures include regular inspection or emissivity alarms to detect bulb failures or film occlusions, regular UV bulb sleeve cleaning and wiping, downstream chlorine detectors, downstream polishing deionizers, and regular (approximately yearly) bulb replacement.

Distillation units provide chemical and microbial purification via thermal vaporization, mist elimination, and water vapor condensation. A variety of designs is available including single effect, multiple effect, and vapor compression. The latter two configurations are normally used in larger systems because of their generating capacity and efficiency. Distilled water systems require different feed water controls than required by membrane systems. For distillation, due consideration must be given to prior removal of hardness and silica impurities that may foul or corrode the heat transfer surfaces as well as prior removal of those impurities that could volatize and condense along with the water vapor. In spite of general perceptions, even the best distillation process cannot afford absolute removal of contaminating ions and endotoxin. Most stills are recognized as being able to accomplish at least a 3 to 4 log reduction in these impurity concentrations. Areas of concern include carry-over of volatile organic impurities such as trihalomethanes (see Source and Feed Water Considerations) and gaseous impurities such as ammonia and carbon dioxide, faulty mist elimination, evaporator flooding, inadequate blowdown, stagnant water in condensers and evaporators, pump and compressor seal design, pinhole evaporator and condenser leaks, and conductivity (quality) variations during start-up and operation.

Methods of control may involve preliminary decarbonation steps to remove both dissolved carbon dioxide and other volatile or noncondensable impurities; reliable mist elimination to minimize feedwater droplet entrainment; visual or automated high water level indication to detect boiler flooding and boil over; use of sanitary pumps and compressors to minimize microbial and lubricant contamination of feedwater and condensate; proper drainage during inactive periods to minimize microbial growth and accumulation of associated endotoxin in boiler water; blow down control to limit the impurity concentration effect in the boiler to manageable levels; on-line conductivity sensing with automated diversion to waste to prevent unacceptable water upon still startup or still malfunction from getting into the finished water distribute system; and periodic integrity testing for pinhole leaks to routinely assure condensate is not compromised by nonvolatized source water contaminants.

Storage tanks are included in water distribution systems to optimize processing equipment capacity. Storage also allows for routine maintenance within the pretreatment train while maintaining continuous supply to meet manufacturing needs. Design and operation considerations are needed to prevent or minimize the development of biofilm, to minimize corrosion, to aid in the use of chemical sanitization of the tanks, and to safeguard mechanical integrity. These considerations may include using closed tanks with smooth interiors, the ability to spray the tank headspace using sprayballs on recirculating loop returns, and the use of heated, jacketed/insulated tanks. This minimizes corrosion and biofilm development and aids in thermal and chemical sanitization. Storage tanks require venting to compensate for the dynamics of changing water levels. This can be accomplished with a properly oriented and heat-traced filter housing fitted with a hydrophobic microbial retentive membrane filter affixed to an atmospheric vent. Alternatively, an automatic membrane-filtered compressed gas blanketing system may be used. In both cases, rupture disks equipped with a rupture alarm device should be used as a further safeguard for the mechanical integrity of the tank. Areas of concern include microbial growth or corrosion due to irregular or incomplete sanitization and microbial contamination from unalarmed rupture disk failures caused by condensate-occluded vent filters.

Distribution system configuration should allow for the continuous flow of water in the piping by means of recirculation. Use of nonrecirculating, dead-end, or one-way systems or system segments should be avoided whenever possible. If not possible, these systems should be periodically flushed and more closely monitored. Experience has shown that continuously recirculated systems are easier to maintain. Pumps should be designed to deliver fully turbulent flow conditions to facilitate thorough heat distribution (for hot water sanitized systems) as well as thorough chemical sanitant distribution. Turbulent flow also appear to either retard the development of biofilms or reduce the tendency of those biofilms to shed bacteria into the water. If redundant pumps are used, they should be configured and used to avoid microbial contamination of the system.

Components and distribution lines should be sloped and fitted with drain points so that the system can be completely drained. In stainless steel distribution systems where the water is circulated at a high temperature, dead legs and low-flow conditions should be avoided, and valved tie-in points should have length-to-diameter ratios of six or less. If constructed of heat tolerant plastic, this ratio should be even less to avoid cool points where biofilm development could occur. In ambient temperature distribution systems, particular care should be exercised to avoid or minimize dead leg ratios of any size and provide for complete drainage. If the system is intended to be steam sanitized, careful sloping and low-point drainage is crucial to condensate removal and sanitization success. If drainage of components or distribution lines is intended as a microbial control strategy, they should also be configured to be completely dried using dry compressed air (or nitrogen if appropriate employee safety measures are used). Drained but still moist surfaces will still support microbial proliferation. Water exiting from the distribution system should not be returned to the system without first passing through all or a portion of the purification train.

The distribution design should include the placement of sampling valves in the storage tank and at other locations, such as in the return line of the recirculating water system. Where feasible, the primary sampling sites for water should be the valves that deliver water to the points of use. Direct connections to processes or auxiliary equipment should be designed to prevent reverse flow into the controlled water system. Hoses and heat exchangers that are attached to points of use in order to deliver water for a particular use must not chemically or microbiologically degrade the water quality. The distribution system should permit sanitization for microorganism control. The system may be continuously operated at sanitizing conditions or sanitized periodically.

Classical culture approaches for microbial testing of water include but are not limited to pour plates, spread plates, membrane filtration, and most probable number (MPN) tests. These methods are generally easy to perform, are less expensive, and provide excellent sample processing throughput. Method sensitivity can be increased via the use of larger sample sizes. This strategy is used in the membrane filtration method. Culture approaches are further defined by the type of medium used in combination with the incubation temperature and duration. This combination should be selected according to the monitoring needs presented by a specific water system as well as its ability to recover the microorganisms of interest: those that could have a detrimental effect on the product or process uses as well as those that reflect the microbial control status of the system.

There are two basic forms of media available for traditional microbiological analysis: “high nutrient” and “low nutrient”. High-nutrient media such as plate count agar (TGYA) and m-HPC agar (formerly m-SPC agar), are intended as general media for the isolation and enumeration of heterotrophic or “copiotrophic” bacteria. Low-nutrient media such as R2A agar and NWRI agar (HPCA), may be beneficial for isolating slow growing “oligotrophic” bacteria and bacteria that require lower levels of nutrients to grow optimally. Often some facultative oligotrophic bacteria are able to grow on high nutrient media and some facultative copiotrophic bacteria are able to grow on low-nutrient media, but this overlap is not complete. Low-nutrient and high-nutrient cultural approaches may be concurrently used, especially during the validation of a water system, as well as periodically thereafter. This concurrent testing could determine if any additional numbers or types of bacteria can be preferentially recovered by one of the approaches. If so, the impact of these additional isolates on system control and the end uses of the water could be assessed. Also, the efficacy of system controls and sanitization on these additional isolates could be assessed.

Duration and temperature of incubation are also critical aspects of a microbiological test method. Classical methodologies using high nutrient media are typically incubated at 30 to 35 for 48 to 72 hours. Because of the flora in certain water systems, incubation at lower temperatures (e.g., 20 to 25) for longer periods (e.g., 5 to 7 days) can recover higher microbial counts when compared to classical methods. Low-nutrient media are designed for these lower temperature and longer incubation conditions (sometimes as long as 14 days to maximize recovery of very slow growing oligotrophs or sanitant injured microorganisms), but even high-nutrient media can sometimes increase their recovery with these longer and cooler incubation conditions. Whether or not a particular system needs to be monitored using high- or low-nutrient media with higher or lower incubation temperatures or shorter or longer incubation times should be determined during or prior to system validation and periodically reassessed as the microbial flora of a new water system gradually establish a steady state relative to its routine maintenance and sanitization procedures. The establishment of a “steady state” can take months or even years and can be perturbed by a change in use patterns, a change in routine and preventative maintenance or sanitization procedures, and frequencies, or any type of system intrusion, such as for component replacement, removal, or addition. The decision to use longer incubation periods should be made after balancing the need for timely information and the type of corrective actions required when an alert or action level is exceeded with the ability to recover the microorganisms of interest.

The advantages gained by incubating for longer times, namely recovery of injured microorganisms, slow growers, or more fastidious microorganisms, should be balanced against the need to have a timely investigation and to take corrective action, as well as the ability of these microorganisms to detrimentally affect products or processes. In no case, however, should incubation at 30 to 35 be less than 48 hours or less than 96 hours at 20 to 25.

Normally, the microorganisms that can thrive in extreme environments are best cultivated in the laboratory using conditions simulating the extreme environments from which they were taken. Therefore, thermophilic bacteria might be able to exist in the extreme environment of hot pharmaceutical water systems, and if so, could only be recovered and cultivated in the laboratory if similar thermal conditions were provided. Thermophilic aquatic microorganisms do exist in nature, but they typically derive their energy for growth from harnessing the energy from sunlight, from oxidation/reduction reactions of elements such as sulfur or iron, or indirectly from other microorganisms that do derive their energy from these processes. Such chemical/nutritional conditions do not exist in high purity water systems, whether ambient or hot. Therefore, it is generally considered pointless to search for thermophiles from hot pharmaceutical water systems owing to their inability to grow there.

The microorganisms that inhabit hot systems tend to be found in much cooler locations within these systems, for example, within use-point heat exchangers or transfer hoses. If this occurs, the kinds of microorganisms recovered are usually of the same types that might be expected from ambient water systems. Therefore, the mesophilic microbial cultivation conditions described later in this chapter are usually adequate for their recovery.

Examples of instrumental approaches include microscopic visual counting techniques (e.g., epifluorescence and immunofluorescence) and similar automated laser scanning approaches and radiometric, impedometric, and biochemically based methodologies. These methods all possess a variety of advantages and disadvantages. Advantages could be their precision and accuracy or their speed of test result availability as compared to the classical cultural approach. In general, instrument approaches often have a shorter lead time for obtaining results, which could facilitate timely system control. This advantage, however, is often counterbalanced by limited sample processing throughput due to extended sample collection time, costly and/or labor-intensive sample processing, or other instrument and sensitivity limitations.

Furthermore, instrumental approaches are typically destructive, precluding subsequent isolate manipulation for characterization purposes. Generally, some form of microbial isolate characterization, if not full identification, may be a required element of water system monitoring. Consequently, culturing approaches have traditionally been preferred over instrumental approaches because they offer a balance of desirable test attributes and post-test capabilities.

The following general methods were originally derived from Standard Methods for the Examination of Water and Wastewater, 17th Edition, American Public Health Association, Washington, DC 20005. Even though this publication has undergone several revisions since its first citation in this chapter, the methods are still considered appropriate for establishing trends in the number of colony-forming units observed in the routine microbiological monitoring of pharmaceutical waters. It is recognized, however, that other combinations of media and incubation time and temperature may occasionally or even consistently result in higher numbers of colony-forming units being observed and/or different species being recovered.

The extended incubation periods that are usually required by some of the alternative methods available offer disadvantages that may outweigh the advantages of the higher counts that may be obtained. The somewhat higher baseline counts that might be observed using alternate cultural conditions would not necessarily have greater utility in detecting an excursion or a trend. In addition, some alternate cultural conditions using low-nutrient media tend to lead to the development of microbial colonies that are much less differentiated in colonial appearance, an attribute that microbiologists rely on when selecting representative microbial types for further characterization. It is also ironical that the nature of some of the slow growers and the extended incubation times needed for their development into visible colonies may also lead to those colonies being largely nonviable, which limits their further characterization and precludes their subculture and identification.

Methodologies that can be suggested as generally satisfactory for monitoring pharmaceutical water systems are as follows. However, it must be noted that these are not referee methods nor are they necessarily optimal for recovering microorganisms from all water systems. The users should determine through experimentation with various approaches which methodologies are best for monitoring their water systems for in-process control and quality control purposes as well as for recovering any contraindicated species they may have specified.