COMMENTARY

Reverse Osmosis as a Means of Water For InjectionProduction: A Response to the Position of the EuropeanMedicines AgencyT. H. MELTZER1,*, R. C. LIVINGSTON2, R. E. MADSEN3, M. W. JORNITZ4, R. M. JOHNSON5,and M. W. MITTELMAN6

1Consultant; Capitola Consultancy, 8103 Hampden Lane, Bethesda, MD 20814; 2CTO (Chief Technical Officer); ArionWater, Inc, 67 Willow Avenue, Hyannis, MA 02601; 3President; The Williamsburg Group, LLC, 18907 LindenhouseRoad, Gaithersburg, MD 20879; 4Group Vice-President; Sartorius Stedim Biotech, 131 Heartland Boulevard,Edgewood, NY 11717; 5Compliance Specialist; Global Manufacturing and Supply, GlaxoSmithKline, 1500 LittletonRoad, Parsipanny, NJ 07054; 6Visiting Scientist; Harvard University School of Engineering & Applied Sciences, 40Oxford Street, Rm. 301, Cambridge, MA 02138 © PDA, Inc. 2009

Introduction

The purpose of this writing is to formalize a reply tothe European Medicines Agency (EMEA) regardingtheir rejection of reverse osmosis (RO) as a suitablemethod for the generation of water for injection(WFI). As stated in EMEA/CHMP/CVMP/QWP/28271/2008, the EMEA preference is for distilla-tion. The EMEA identify several areas of the ROmethod they consider objectionable with regard toits microbiological aspects. These include biofilmwith its implications of ever-present organisms.Statements made include, “Biofilms cannot be de-stroyed”; also, “The biofilm will build up and be-come increasingly resistant to sanitisations by hotwater or by chemicals because of the glycocalyxmaterial.”

The permeability of the RO filters is seen as beingunsafe. The EMEA document states, “a range of met-abolic byproducts will be secreted which will includeproteins and carbohydrates, some of which may bebiologically active. These contaminants are not easilyidentified or quantified.” Endotoxins share in this con-cern.

In summarizing their objections to the RO process,EMEA state, “Today it is not possible to assumethat the quality of WFI prepared by reverse osmosis

is as safe as WFI prepared by distillation accordingto the requirements of the European Pharmaco-poeia.”

This statement, sincerely held, offers no room fordiscussion. The EMEA have the right to adopt theirown standards. There is ample evidence, however, thatthe EMEA share our earnest desire for cooperation andan exchange of views. The purpose of this rejoinder isto address the stated EMEA concerns in the hope offurthering harmonization.

Sterility of WFI

A possible inexactness in the terms used in thewater purification processes should be eliminated.The WFI referred to by the United States Pharma-copeia (USP)—and the European Pharmacopoeiaand the US Food and Drug Administration(FDA)—is a bulk water; it is not sterile, as isindicated by its numerical microbial limits. Paren-teral preparations formulated using this WFI arerendered sterile by steam autoclaving or sterilizingfiltration subsequent to their being compounded.The heat involved kills the predominant Gram-neg-ative bacterial population and thereby generates en-dotoxins. That the actual microbial content of thebulk WFI was not excessive is assured by the en-dotoxin limit set by the authorities for the (small)volume parenterals, namely, 0.25 EU/mL. In effect,the endotoxin limit serves as a guarantor for thepropriety of the bulk WFI’s organism content. Al-ternatively, the preparations are subjected to filtra-tion sterilizations using a microporous “sterilizing

*Corresponding author: Dr. Theodore H. Meltzer, Tel:301.986.8640; E-mail: theodorehmeltzer@hotmail.com

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filter” as defined by the FDA. This procedure, unlikethe employment of RO filters, includes the integritytesting of the filters and of the filtration process.

Where parenterals are compounded without the ben-efit of autoclaving, as when proteins or other heat-sensitive components are involved, filtrative steril-izations are relied upon. In such exercises,validation is relied upon to ensure that a sterileproduct results. Validation is not a “best effort”exercise; it is a documented, experimentally dem-onstrated performance indicating that within thebounds of acceptable risk the process is dependableand can reliably be replicated. The point being madeis that WFI is not sterile WFI (SWFI) (1) and is nota substitute for it. To gain the appellation of “ster-ile,” the SWFI is produced by methods validated toimpart that quality.

Regarding the EMEA Summary

In summary the EMEA state, “it is not possible toassume that the quality of WFI prepared by reverseosmosis is as safe as WFI prepared by distillation.”The operative word is “assume.” There is neither thejustification nor the need for the assumption. Relianceshould derive from documented experimental evi-dence, from validation of the process. One may chooseto question the value of the validation concept or thethoroughness of its performance. But the implicationsof “assumptions” are needlessly pejorative. The ROusage for WFI purposes has many times been vali-dated, and probably is every day.

One understands that the EMEA are well aware of thequality requirements of the distillation and RO feedwaters, and of the need to preserve the qualities of theproduct waters attained by application of the respec-tive process. Be that as it may, the complete absenceof any references to the absolute necessity for suchpre-treatments and post-treatments invites an over-reliance on the performance of these processes, as ifthey were innately free from error. For example, chro-matographic applications are so sensitive to biofilmsthat the protection of multiple treatment steps is reliedupon to limit the possibilities of their formation. Ap-propriate cleaning and sanitization regimens, includ-ing sterilizing-grade filtrations are validated for thisvery purpose. It is the maintenance of the feedstream’s essential quality that is the key to the properperformance of the RO or distillation operation. Nei-

ther distillation nor RO is the complete show. Thelimitations of each are amenable to compensatory cor-rections.

One would be better served by the statement that thefeed streams for both RO and distillation could be thedetermining factor for the quality of the final results. Ifthe feed stream is contaminated in front of the distil-lation column, the quality of the resulting WFI isquestionable; similarly so with RO. It is not only thetechnology generating the WFI, but the performanceand maintenance of the entire purification train that aredecisive.

With regard to the metabolic by-products and thedifficulty of their identification, it is fair to say that theEMEA’s invoking the possible but not the certain, andthe conceivably-present but not the identifiable, leavesno margin for discussion. As said at the start of thiswriting, the EMEA are entitled to the standards andconsiderations they consider appropriate. If, however,there is to be discussion, the topics must be specificenough to characterize with some exactness. Other-wise, refutation cannot be enjoined.

Identical Microbiological and Chemical Quality

In partial reply to the above, it can be said that theEMEA standards for WFI prepared by distillation andthe standards set by USP for WFI prepared by RO areidentical with regard to their microbiological andchemical contents. If so, the EMEA statement can bedisputed. In this matter, then, it is the means of prep-aration that are in question, not “the quality of theWFI” that is in dispute.

Differences in Means of Preparation

The heat associated with the distillation process ad-dresses the organism problem by killing the microbes.RO operates to remove them from the WFI by filtra-tion. Few will argue that the finality of death occa-sioned by heat is not more dependable in eliminatingorganisms than is their filtrative removal. If, however,the standards of the end products are the same and aremet, perhaps the potential advantage of the heat fur-nished by distillation is not a necessary condition tothe preparation of WFI. If heat is an essential factor itcan be furnished by use of hot RO systems.

It is not being advocated that the means of preparationare suitable if the stipulated product quality is

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achieved. The need for reliance upon validated pro-cesses is recognized. The aphorism that “Qualityshould not be tested into the product” is not contested.One may see dogmatic steadfastness to this maxim asthe upholding of principle. However, in our commend-able efforts to minimize risk, we should be careful notto create the “irresistible force” meeting the “immov-able object.” However challenged by the EMEA, it isa fact that the RO process is a validated operation.

Distillation Concerns

The EMEA document lists limitations of the ROmethod used in the preparation of WFI. Perhaps asimilar listing with regard to the distillation processwould be proper. Such would seem to be necessarywhere a comparison of different methods is beingrelied upon in making a choice.

Stills vaporize liquid water. The vaporization of wateras a means of separating it from its solutes or sus-pended particles is a useful method of purification. Itpermits the separation of the water in its gaseous formfrom its non-volatile impurities. The water vapor isthen condensed into its liquid state, enabling it to beseparated from its non-condensable, volatile impuri-ties. However, such neat distinctions need not neces-sarily be obtained. Because of the heat involved, dis-tillation can be a self-sanitizing system. Unfortunately,the need for stills to expeditiously furnish large vol-umes of distillate necessitates high heat inputs thatprovide opportunities for the vapor entrainment offeed water droplets. A consequence is the carry-overwithin the droplets of non-volatile impurities. Thedistillation requires careful implementation to avoidsuch entrainments. The carry-over of impurities mayresult also from a foaming of the waters in the still pot.Such may be caused by given organic compounds,total organic carbon (TOC). Departures from the ideal,as also with the RO technique, can occur.

Droplet carry-over, the entrainment of liquid watercum contaminants, is minimized by the still design,but is possible nonetheless. Its avoidance, involvingblowdowns and heat inputs, compels an active andeffective management.

Not all substances can be separated by the water’sbeing vaporized. TOC may be a problem. Certainorganic compounds will co-distill as pollutants whenazeotropic combinations are present, even when the

distillation is operated in an optimal fashion. Indeed,steam-distillation provides a method, used in organicchemical productions, to isolate from a mix of mate-rials substances that would otherwise require too higha temperature to be distilled. Depending upon its vaporpressure, the organic chemical will co-distill and be-come condensed as an ingredient of the distillate.

A very proper operation of the still is needed to avoidunwanted occurrences. The validation of the still op-eration, therefore, involves documented experimentalevidence that the standard operating procedure (SOP)protocols appropriate for the still were adhered to.Still manufacturers will specify the purity of the feedwaters to be supplied their stills. Evidence will berequired attesting to the conformity with such stipu-lations. The point being made is that for the properresults to eventuate from distillations, as also from ROsystems, a specified feed water quality is requiredalong with a correct implementation. The distillingoperation is not an automatic activity free of demand-ing requirements. It bears a strong burden of exacti-tudes.

Distillation and Endotoxin Carryover

A major concern in still operations is the possibility ofendotoxins from the feed waters being vapor-entrainedinto the condenser. Still operations are not absolute.Rather they are depended upon to provide log reduc-tions of impurities. Ideal distillation may be capable ofproviding better results than may routinely be ob-tained. However, fabricators of stills are reluctant toguarantee that still operators will accomplish endo-toxin reductions greater than three or four logs. Themanufacturers of stills do not question the intrinsiccapabilities of their instruments to do better. Presum-ably, on the basis of experience, they rather expect thedistilling operations not to be managed with the ex-pertness necessary to attain the higher results that mayinherently be possible.

The FDA requires, therefore, that the endotoxin con-tent of the feed waters not exceed concentrationsgreater than those whose management by distillationcan be assured by the still makers. Whatever qualityparameters are set in the SOPs relevant to the feedwaters and to the operational manipulations of the stillmust, as part of the validation exercise, prevail inpractice. This establishes maximum endotoxin limits

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for feed water contents. The FDA requires and expectsvalidations to be performed.

Metallic Impurities

Metallic entities may also be problematic in distilla-tions. Stills are usually constructed of austenitic steelsbecause of their receptivity to passivation, a processthat prevents the generation of rusts. This necessitatesthe addition of certain quantities of chromium, nickel,and (usually) molybdenum to the steel. The presenceof these metals in ionic forms or as carbonates, car-bides, or hydrated oxides is an inevitable consequence,albeit small in quantities. In applications such as thepreparation of semiconductor rinse waters where me-tallic compounds are anathema, steels, austenitic or ofother compositions, are avoided. So inevitable is thepresence of metallic compounds in the waters preparedusing stills that a particular term is used in referring toits occurrence, namely, the metal signature.

Some quantitation of the metallic impurities may be inorder. In the preparation of high-purity waters forelectronic usage, the employment of the austeniticsteels and their necessary passivation are avoided. Thestandards for the metallic ion contents of semiconduc-tor rinse waters set by the ASTM D-5127-99 for typeE-1 items with line widths of 1.0 – 0.5 are 0.05 �g/L(0.05 ppb) for iron, copper, chromium, aluminum,lead, nickel, manganese, zinc, etc. These values areattained using RO and avoiding distillation.

The high standards established for metals intended forelectronic waters also characterize the limits set forbacteria, endotoxins, and water resistivity. These arealso expected to be forthcoming from RO operations.Bacteria are limited to 1 CFU per 100 mL, and as strictas 1 CFU per 1 L, according the Balazs UltrapureGuidelines, whether dead or alive. The limits on thenumber of on-line particles per given micron rangesizes are 500/0.05– 0.1, 300/0.1– 0.2, 20/0.2– 0.3, 20/0.3– 0.5, and 4/�0.5. The resistivity of the water at 25°C is 18.2 microsiemens, �S-cm2; the endotoxin con-tent is set at 0.03 EU/mL; the TOC is limited to �5�g/L, or �5 ppb.

Such high standards are not needed for pharmaceuticalpurpose, nor are they being proposed, but they areevery day attained by the use of RO equipment. Thedistillation process may merit the superiority theEMEA assigns it, but the RO may be eminently suit-able nonetheless.

Metallics and Pharmaceuticals

There are also strict limits on heavy metals in watersintended for certain biotechnology products. For ex-ample, monoclonal antibodies and certain other pro-tein-based items can be very sensitive to trace levels ofthe transition metals, such as nickel, chromium, etc.,that may leach form stainless steels. Significantly,WFI meeting compendial requirements, other thanconductivity and heavy metals, may not be suitable forsome of these products. As in the case of microelec-tronic usage, some biotechnology-based pharmaceuti-cal materials may require waters that have been pre-pared in non-metallic systems. By necessity, therefore,distillation may be precluded. RO may then be theonly means available for producing WFI.

The Purification System

It is more than wordplay to say that WFI is notprepared by either distillation or by RO alone, but bysystems that include one of these two methods. Aconsiderable pre-treatment regimen may be needed toprepare source waters for distillation or RO. Withoutsuch, distillation or RO may be of limited worth. Thepreparation of WFI utilizing distillation or RO is notan end in itself. However prepared, the quality of thewater must be maintained throughout the storage anddistribution steps. The involvement of distillation orRO, however important, is only one necessary actionin preparing WFI. Waters of WFI quality are prepareddaily in large quantities using RO for the electronicand semiconductor operations, but never by RO alone.In semiconductor water purification, the RO is a re-quired process, but only one important process amongothers, such as TOC reducing UV, mixed bed deion-ization and sub-micron filtration to assure that semi-conductor water quality is achieved. The misuse ofeither method is possible; each has its limitations. Ofobvious concern is the sterility of the prepared waters.It requires being preserved in that condition.

Cold storage and distribution systems have the poten-tial for microbial growth and for the formation ofbiofilm. Parts of the latter, as the EMEA takes pains toemphasize, undergo shedding to contaminate thestored water with the microbes and their metabolicproducts. The need for periodic and timely antimicro-bial treatments (sometimes referred to as “sanitiza-tions”) of the water storage and distribution loop isindispensable. Regardless of the initial sterility of the

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distilled water, or the colony-forming unit (CFU)counts of normally produced RO waters, the WFIsystem will almost assuredly require a regimen ofintermittent sanitizations. Storage under ozone, or atelevated temperatures at �65– 85 °C is usually re-sorted to. Curiously, the EMEA document makes nomention of heated RO systems. Operated and main-tained at �65– 85 °C, the hot-water installations havebeen shown to be bacteria-free.

Microbial Recontamination

The EMEA paper states, “The reverse osmosis devicemust be validated to prepare a quality of water iden-tical with water prepared by distillation.” If this state-ment refers to the system using RO or distillation, it iscorrect. But that is already the case. If the statementmeans only the RO or distillation purification unit perse, may one ask why? Will sterility, once achieved,remain inviolate?

Even if initially sterile, the distilled water stored overtime may become contaminated with microorganisms.Such can invade the system through the vent filtersmeant to protect it from microbe-contaminated air,from improper sampling activities, from defects in theRO membranes, from distillation carry-over, and fromopenings of the system for inspections, etc. The lim-itless continuance of the sterile condition dependsupon defenses that may prove imperfect, and uponmanipulations that may, on occasions, be improper.One will not encounter in the manufacture of sterilewaters that degree of perfection that eludes mankind inall of its other pursuits. With regard to WFI, its storageunder ozone or in a heated condition can preserve it ina bacteria-free state and maintain it as such. However,sanitizations may be required from time to time evenof such systems.

Organism Limits of Purified Water

The initial sterility of the distilled water will availlittle if it is not protected. If, on the other hand, therelatively few organism counts in the RO water areseen as a marked encouragement to microbialgrowth, the question may be asked as to why theEMEA does not stipulate distillation for PurifiedWater preparation as well. Consider, the chief con-cern regarding WFI is its bioburden content. Thepermissible limit is 10 CFU per 100 mL. It isunderstood that the actual microbial count may vary

by one-half log or so. The bioburden allowed forPurified Water is set at a higher limit, namely, 100CFU per mL, because its applications are for lesscritical usages—perhaps less critical, but criticalnonetheless. The pseudomonads and related Gram-negative bacteria, common to waters, are with someexceptions not regarded as being particularly dan-gerous. There are specific interdictions againstPseudomona aeruginosa, know to cause skin infec-tions, and against Pseudomona cepacia, now namedBurkholderia cepacia, also known to cause harmfuleffects. Pseudomona fluorescens has been impli-cated in encephalitic incidents. There is, therefore,reason to believe that the pseudomonads and relatedGram-negative bacteria, being opportunistic patho-gens, are not inherently benign. If distillation is soreliably destructive of organisms, one wonders whythe EMEA does not advocate its application also tothe preparation of purified water. An EMEA list ofpurported shortcomings of the RO method includesthe passage of organisms in the form of biofilmduring the “flexing and bending” of the RO mem-branes. Nonetheless, this very risk of organism con-tamination is countenanced by the EMEA in thepreparation of purified water. Why? Perhaps, de-spite all that is said, there is the subliminal recog-nition that numerous facilities have operated ROsystems for purified water trouble-free for years,with proper maintenance and control.

Biofilm

Of significance is that the water prepared by distilla-tion or RO must retain its “use quality” in the storageand/or distribution section of the system. It is in thisambience that considerations of biofilm formation areimportant. However prepared, the product waters willnot be exempt from biofilm formation; this includesdistillation. Biofilm is an inevitable occurrence in allwater systems except those continuously maintainedhot, at above at least 65 °C, or (stored) continuouslyunder an ozone umbrella. Very rarely is the presenceof biofilm detected by opening the system and remov-ing test sections installed for the very purpose ofinspection. Otherwise, one relies upon the microbio-logical assaying of the water. Biofilm sloughing ismade evident from oscillating cell counts. Sanitiza-tions may then be instituted. They may be performedprophylactically at periods made evident during thesystem’s validation.

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EMEA References to Biofilms

Some of the EMEA negatives concerning biofilms areperhaps somewhat overstated. Regardless, the EMEAremarks about biofilm are serious and merit concern:

● Biofilm formation on both sides of the RO mem-brane begins within minutes of the unit beingswitched on.

● Biofouling of membranes will begin within a fewminutes of operation of the system.

How readily biofilm forms depends to some degreeupon the singularity of the particular system. Thespeed of organism attachment to the RO filter andbiofilm development is stated by EMEA to require but“a few minutes.” However, the speed is irrelevantunless the mention of it by the EMEA is meant tosuggest that control of biofilm is quite impossiblebecause the speed of its reasserting itself renders itssanitization futile. The facts are otherwise. The manage-ment of biofilm formation and its sloughing of organismscan be and is contained and managed routinely.

The EMEA document states

● “RO is a percentage removal system: the higherthe concentration of the micro-organisms, themore metabolic by-products, endotoxins, etc. willpass through the membrane.”

● “The net effect is that the RO membrane willbecome, in practice, a bacterial fermenter. As bac-teria in the biofilm grow and metabolise, a range ofmetabolic by-products will be secreted which willcontain proteins and carbohydrates, some of whichmay be biologically active. These contaminantsare not easily identified and quantified.”

There are no data that support the EMEA contentionthat biofilms form on feed- and product-water surfacesof properly operating RO systems within minutes.Certainly, bacteria can attach to membrane surfaces onthe feed-water side. Appropriate chemical treatmentregimes developed as part of the validation processprevent fouling and associated flux declines. Signifi-cantly, bacteria cannot “grow through” RO mem-branes, which are permeable only to some ions andwater molecules, both of which are orders of magni-tude smaller than bacteria (or viruses).

Similarly, the contention that by-products of bacterialmetabolism (e.g., endotoxins and other organic com-pounds) can somehow be generated on membranesurfaces then permeate a properly functioning ROsystem is simply not supported by the scientific evi-dence. The minimum MW (molecular weight as anindex of molecular size) for the lipid-A portion ofendotoxins (required to elicit a fever response) is on theorder of 10,000; the approximate organic molecularweight cutoff for RO membranes is on the order of 300.

The statement that biofilms will become increasinglyresistant to sanitization by hot water or chemicals isuntrue. While microbial biofilms can provide a differ-ential resistance to chemical and physical treatmentagents, this resistance is associated with much lowertemperatures and chemical concentrations than areused in industrial practice. Biocides are not the sameas antibiotics. Mesotrophic bacteria, such as those thatpredominate in pharmaceutical water systems, arekilled by water temperatures exceeding approximately50 °C, and there is certainly no evidence of a consti-tutive or induced heat resistance in these organisms.

Similarly, the statement that biofilms cannot be de-stroyed is also incorrect. Commonly employed ROtreatment agents such as peracetic acid, hydrogen per-oxide, and peracetic acid/hydrogen peroxide combina-tions are sporocidal and hydrolyze biofilms (includingendotoxins).

The EMEA document states

● “The biofilm will build up and become increas-ingly resistant to sanitization by hot water orchemicals because of the glycocalyx material.”

● “Biofilms cannot be destroyed. Any attempts byusing biocides, temperature, etc. results in a rapidincrease in growth following treatment.”

These statements are not correct. They are in thenature of painting stripes on a tiger.

RO Mechanical Properties

The EMEA paper sets forth the potential for flaws andweaknesses in the mechanical properties of the ROmembranes:

“Biofilm can form on the permeate side of the mem-brane in the following ways:

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● Through microscopic holes and tears in the mem-brane.

● Through flexing and bending as pressure increasesdue to decreasing flux as the biofilm builds up.”

With regard to the mechanical strength of RO mem-branes, mention is made of ceramic filters. Perhaps theencouragement of possible future efforts, namely, theforthcoming of ceramic filters, is intended to demon-strate an open-mindedness towards filtration despitethe seeming intransigence on the present type ROfilters.

In reference to filter robustness, there have been de-velopments in polymeric membrane design and con-struction following the original position paper oppos-ing the RO process for WFI production. If an accountof these developments could be of help in moving thisdiscussion further, the authors would be pleased tosupply same in detail.

Another question needs to be asked. The EMEA doc-ument states, “The reverse osmosis device must bevalidated to prepare a quality of water identical withwater prepared by distillation. It appears that manydevices available on the market are not designed forsuch a use.”

But are not the inadequacies of such devices detectedduring the required validation exercise? Surely, rele-vant regulations prohibit their usage in processingapplications unless their validations are successfullyperformed. If the acceptability of a validated device isvitiated by the failure of others of its kind, is thejustification of such rejections established on any sta-tistical or other objective basis?

Towards Harmonization

The path to harmonization might lead through therecognition that the “WFI” product as prepared byeither method is the equivalent of the other withrespect to its indispensable qualities. Within thebounds of acceptable risk, qualities of lesser perti-nence to the WFI application may be accorded asubsidiary concern. The particular influences of thefeed-water preparation, and the storage and distribu-tion of the product water of the individual system,should be assessed and evaluated with regard to the

suitability of RO, distillation, or any other method forWFI preparation.

What is now necessary, we believe, is to ascertainwhether the EMEA’s reconsideration of the use ofmethods, other than distillation, presently available forthe manufacture of WFI is at all possible.

Reference

1. Baszkin, A.; Lyman, D. J.; Meltzer, T. H. Theoret-ical considerations of bubble point measurement:solid/liquid wetting interaction. Pharm. Technol.Int. 1979, 2, (l), 22–31.

Ancillary Sources Regarding Biofilms

Block, S. S. Disinfection, Sterilization, and Preserva-tion. Lippincott Williams & Wilkins: Malvern, PA,2001; p 1481.

Gorke, A.; Kittel, J. Routine disinfection of the totaldialysis fluid system. EDTNA EDTA J. 2002, J28 (3),130 –133.

Marshall, K. C. Biofilms: an overview of bacterialadhesion, activity, and control at surfaces. AmericanSociety for Microbiology News 1992, 58 (4), 205.

Mittelman, M. W. Bacterial Biofilms in Pharmaceuti-cal Water Systems. In Filtration in the Biopharmaceu-tical Industry; Meltzer, T. H., Jornitz, M. W. Eds.;Marcel Dekker: New York, 1998; pp 245–258.

Mittelman, M. W. Structure and functional character-istics of bacterial biofilms in fluid processing opera-tions. J. Dairy Sci. 1998, 81, 2760 –2764.

Mittelman, M. W.; White, D. C. The Role of Biofilmsin Bacterial Penetration of Microporous Membranes.Proceedings of the Pharmaceutical Technology Meet-ings: Philadelphia, PA, 1989; pp 211–221.

Mittelman, M. W.; White, D. C. The Role of Biofilmsin Contamination of Process Fluids by Biological Par-ticulates. In Particles in Gases and Liquids; Mittal,K. L., Ed.; Plenum Press: New York, 1990; Vol. 2, pp33–50.

Ridgway, H. F.; Rigby, M. G.; Argo, D. G.. Bacterialadhesion and fouling of reverse osmosis membranes.J. Am. Water Works Assoc. 1985, 77, 97–106.

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