Theodore H. Meltzer is principle of Capitola Consultancy.

Theodore H. Meltzer

Principles and Modern Applications of Mass Transfer Operations, 

has taught this subject in the chemical engineering department of the University of Puerto Rico in Myagüez for some three decades. Before he began work on the first edition, Benítez was determined to write a textbook, not just a text that would be used with students simply because it was the only book to deal with a specialized subject, according to biographical information included in the book. A textbook should be written by an educator who can impart his understanding to others. The author should not only be an expert in the subject, but should also be able to communicate to students the logic of the discipline, its causes and effects, and how it can be applied practically to solve problems. 

Benítez's challenges are to present the problem of mass transfer in its full complexity to the novice, to parse the relationships between its components, and to express the subject in the language of mathematics to solve problems. Providing such a chain of logic requires far more than an expert knowledge of the subject, but Benítez is equal to the task. His ability to communicate with the uninitiated is awesome. 

For each of the numerous examples he uses to demonstrate mass-transfer applications, he sets forth the problem, provides the necessary data, explains the underlying physical laws, presents relevant experimental references, and indicates the mathematical path to the solution. The text includes too many examples of this approach to generalize, but each instance is impressive in its relevance and practicality.  

Principles and Modern Applications of Mass Transfer Operations by Jaime BenÃ­tez, Wiley, Hoboken, NJ, 2009, 620 pp., ISBN: 0470181788 

Benítez states that the methods of mass-transfer operations include such techniques as distillation, gas absorption, adsorption, humidification, liquid extraction, and membrane separations. Benítez incorporates Mathcad, software that expresses problems in mathematical notation and verifies engineering calculations, for example problems and homework. An appendix lists many such programs, which can help solve Maxwell–Stefan equations, perform packed-column calculations, assist sieve-tray design, solve binary distillation problems, and perform multistage crosscurrent extractions. 

Mass-transfer operations is not an everyday subject. Even today, it remains largely the province of the chemical engineer. But in this reviewer's opinion, the subject should be a required course in every physical-science curriculum today because it is basic to almost all separation processes. Mass transfer is related to the tendency toward equilibrium that derives from concentration gradients. It involves the transfer of material, or mass, from one homogeneous phase to another. Any arrangement that includes several components in unequal concentrations exhibits mass transfer toward equilibrium (i.e., from the concentrate to the dilute). 

The text's breadth of focus is astonishing. It deals with interphase mass transfer and the relevant theories, the material balances, the equilibria, and equilibrium-stage operations. It treats diffusion, diffusion coefficients, and diffusion in solids and liquids. Other subjects covered include the distillation of multicomponents, batch distillations, packed and trayed towers, Fenske's equation, and optimum-reflux ratios. Among many other activities discussed in similar detail are humidification operations, mass transfer in membranes, and sorption processes. 

Regarding membrane separation techniques, Benítez discusses such current processes as dialysis, reverse osmosis, microfiltration, and ultrafiltration, as well as gas permeation. This reviewer, as must be apparent, considers this book to be a most worthwhile addition to the technical literature. 

Today's students are lucky to have textbooks such as Benítez's for their instruction. Mass-transfer operations were little organized as a discipline in the 1930s and '40s. Scientists formerly used distillation and liquid-extraction techniques, prepared ultrapure water, and purified drugs with membrane processes without sufficient instruction in mass-transfer operations. Only recently has industry realized the importance of the hydrogen bond, understood the reflux ratio in distillations, and appreciated the centrality of surface physics. 

Rather than learning by trial and error, today's students have the means to learn the factors and interactions involved in mass-transfer processes, thus gaining confidence and skill. Understanding is the indispensible basis of practice, and Benítez supplies that understanding up front. 

Benítez also manages to bring a personal and poetic touch to what could have been a dry discussion of chemical engineering. For example, Benítez's example of mass transfer is "the invigorating aroma of a cup of freshly brewed coffee and the sensuous scent of a delicate perfume both reaching our nostrils from the source by diffusion through the air." The reader cannot help but notice and admire the author's perception. 

Through the book's dedication ("To Jaime for being a most special son; your angelic smile is all that is necessary for my happiness."), Benítez reveals his pleasure in dealing with people. His audience, the readers of his text, will be his beneficiaries. 

is a principal of Capitola Consulting, 8103 Hampden Ln., Bethesda, MD 20814-1244, tel. 301.986.8640, fax 301.986.9085, 

Articles

A comprehensive book about mass transfer benefits from the author's personal touch.

A Mass Transfer of Knowledge

Aseptic processing is of great importance within the biotechnology industry because cold sterilization by membrane filtration is the only method of sterilizing biological solutions. Any heat treatment would destructively affect the drug product and target protein. To ensure sterile filtered products maintain their sterile state, an increasing number of companies are using disposable process solutions to process or store the resulting filtrate. Modern disposable connectivity allows joining the filtrate hold bag with the filling line. If the product must be transported to another facility, then the filtrate could be frozen and thawed after it reaches the filling facility. A controlled freeze–thaw process ensures protein degradation is kept to a minimum as a result of the lack of fluid movements and/or uneven protein concentration within the frozen volume. 

In addition, biologics are commonly difficult to remove from fixed, stainless-steel equipment. The cleaning and cleaning qualification process is tedious and must be exceptionally thorough to prevent residues and potential cross-contamination. The cleaning and sterilization down-times for stainless steel vessels, transfer lines, or filter housings might require 8–10 hours and copious amounts of cleaning solutions and water-for-injection (WFI). Calculations have shown that the cleaning and sterilization process of a 1000-L vessel can cost as much as $5000, depending on the WFI costs, whereby a bag of this size is priced at $200–300. The need for shorter cycle times and reduced process-material pushed the development of disposable solutions, which require no set-up times or cleaning. Disposable mixing systems can be connected to capsule membrane filters and a hold bag. These interconnected disposable systems are gamma sterilized and ready to use.  

Another benefit of disposable equipment is the fact that the end-user does not come in contact with the product. Therefore, disposable equipment has increasing popularity in cytotoxic drug applications. The drug product is processed from one disposable process step to the next without the need to either dismantle a filter from a filter housing or clean a vessel by hand. Any time a drug product has a potential to harm the user, disposable processes are the equipment of choice.  

The first disposable units were probably filter capsule devices, which could filter small volumes without the need of a filter housing and required cleaning. These filter capsules incorporated a plastic filter housing and filter cartridge into a one-piece disposable unit (see Figure 1). Capsules nowadays are available in sizes from 100 cm

. These capsules can be assembled to other equipment and autoclaved, or the capsules are delivered presterilized by the filter manufacturer, including equipment connections (e.g., to a filtrate hold bag).  

Figure 1: Small-scale filter capsule devices. (ALL PHOTOS ARE COURTESY OF THE AUTHORS.)			

The next development was single-use sterile bags to replace glass bottles, plastic carboys, or stainless-steel containers for small-volume storage, transport of biological solutions, and growth media. Originally, blood and parenteral solution bags were adapted for process use. Because the mechanical stability had to be strengthened, specific polymeric bag films were developed. These films are laminated in various configurations to gain the mechanical strength for large-volume bags, to reduce any leachable release into the product, and to create an oxygen barrier (see Figure 2).  

The laminate combinations can vary depending on the bag design, manufacturer, and application served. Currently, bag designs are manifold and vary in volume from 20 mL to 3000 L. The assemblies are not restricted to only a bag and a filter. They can be highly complex and engineered to fit specific requirements of the end-user. For example, buffer hold bag systems are commonly a manifold of multiple bags of the same volume, which can be disconnected by tube sealers. These assemblies also contain a sample bag for quality assurance purposes. The filtered volumes can be stored or transferred to individual process steps within a facility. Large-volume systems require storage with trays, tote, or pallet systems to prevent the large liquid volume from moving. The bags within such storage systems require unfolding in a very specific manner, otherwise pressure folds will occur and could damage the polymeric film. The design of the filling and connectivity of any outlet from the bag are essential because the hold-up volume of a hold bag must be minimal.  

As mentioned, disposable systems are no longer restricted to a bag and a filter. Various equipment components are available that can be combined into a single-use system or process step. Such equipment components include: 					

Mixing systems (various designs and volumes)

Bioreactors (many designs, volumes, and agitation technologies) 

Membrane chromatography (e.g., ion exchange)

Viral clearance (e.g., filters and UV inactivation)

Other equipment solutions will soon follow as the requirements for single-use systems by the industry are more pressing and innovative. For example, work is underway to design disposable valves, pump heads, and filling systems. 

Disposable mixing systems are available from 5 to 1000 L and are most commonly used for buffer and media mixing, product compounding, or final formulation purposes. A more recent application is viral inactivation by pH shift. Such mixing bags use disposable sensor patches to determine the necessary pH levels during the inactivation period.  

Mixing methods include recirculation, pulsation of the mixing bag; use of magnetic impellers, stirrer bars, or pads; and use of a levitation mixer. The latter is unique to all others because the mixer does not come in contact with the bag material, and it floats on a magnetic field. This configuration avoids any friction of the bag material and provides fast and thorough mixing results. The various mixing technologies are useful because the applications and complexities of mixing vary. Liquid–liquid mixing has easy mixing requirements, but liquid–powder mixing can be difficult and may require careful design and mixing mode observations.  

Current single-use bioreactor systems are divided into two main categories: rocking motion and cylindrical-tank reactors with various agitation modes. Rocking motion reactors were the first to enter the biopharmaceutical industry. In this design, the cell culture is moved back and forth by a rocking platform, which allows mixing of nutrients and gas input into the liquid phase. These systems produce excellent results for shear-sensitive cell cultures and have been especially established in seed reactor applications. Rocking systems are available as a basic system without sophisticated controls. Separate systems can be connected to a control unit to measure the dissolved oxygen and pH by means of a disposable sensor. The control system can also run the gas and feed strategies, which is important when the system is used as a perfusion reactor. The working volumes of rocking reactors range from 0.2 to 500 L. Recent bioreactor developments have focused on cylindrical tank systems, which use multiple agitation methods, including sleeved or fully polymeric stirrers, vibro-mixing, orbital shaking, or gas mixing. The agitation methods and cell culture requirements determine the design for these reactors. Typical capacity volumes of these cylindrical disposable bioreactor systems range from 10 to 2000 L. These units are most commonly used with the control tower systems of reusable fermentation systems. These control towers create appropriate feed and gassing environments for the cell culture.  

Bulk raw materials are often shipped over long distances, and production processes may require a product-hold step because of downstream equipment bottlenecks. In both instances. solutions are required to prevent any protein degradation, which may occur as a result of enzymatic attack and temperature, pH, concentration, or gas conditions within the hold or transport step. To avoid such yield losses, the industry has resolved to freeze steps to keep the product stabile over a period of time. However, commonly used blast-freeze steps are uncontrolled and can result in freeze concentration, pH shifts, aggregation, ice crystals within the frozen material, or bag damages. To avoid such damaging conditions, disposable controlled freeze–thaw devices have been established. These devices contain a hold bag within a frame and a freeze–thaw module that uses heat-exchanger plates, which ensure a unified controlled freeze and thaw process. The product hold bag with the frame is transferred into the freeze–thaw module, and the heat-exchanger plates move in position, pressing against the hold bag. The heat exchanger plates therefore do not only ensure the temperature transfer, but also the uniform distribution of the liquid over the entire bag design. These units are available as small-scale trial devices with a volume of 30 mL and process scale up to 16 L. 

During the past 10 years, disposable technology innovations surfaced rapidly, and the nondisposable gaps within a biopharmaceutical process are now closing. There are still process steps or specific equipment parts that either will evolve into disposable systems or will be kept and integrated into a hybrid state. How total processes will look like in the future are only predictions, but developments of new disposable process components show the trend toward a total disposable process, at least in the small-scale volume streams (see Figure 3.)  

Figure 3: Possible disposable process stream.			

Such a process would require careful planning because the volumes within a process can change drastically, process steps require proper timing, and essential connectivity requires being qualified. In some instances, radiofrequency identification (RFID) can be used to either to track equipment or process units or to connect the required process units together. These tags will can also create appropriate and necessary shelf-life information, because gamma-irradiated polymers have limited shelf lives.  

The major items still not available as disposable units  are valves and filling lines, which are able to handle fluid volumes at high speeds. There have been attempts to design filling systems, but these have not penetrated the industry as much as the above described disposable technologies. It is only a matter of time when such disposable filling equipment will be made available.  

Innovative developments in disposable equipment enhance the safety in aseptic processing. Furthermore, these developments might create the possibility of a disposable factory. The benefits of complete disposability are relevant to all scales of bioprocesses, especially within the start-up phase. In early development, disposability reduces the need for major capital investments. As these technologies continue to develop critical factors such as drug cost, production cycle times, new product development time, and facility flexibility, all aseptic processing will be affected. 

Because most disposable devices are gamma irradiated, between 25 and 50 kGy short- and long-term, stability studies with the irradiated devices must be performed. Irradiation typically reduces the shelf life of such devices, and it must be determined what the limits are. Furthermore, the irradiation step could accelerate the degradation of the polymeric substances used, which can result in increased leachable and extractable levels. To determine the effects of irradiation and the stability of the polymer used, manufacturers subject these devices to a considerable regime of qualification tests before the device is commercialized. The qualification tests serve as a guidance by the end-user and commonly encompass, but are not limited to, the following tests: 					

Mechanical properties (tensile strength, elongation at break, seal strength, air leak test)

Gas transmission properties (ASTM D3985: oxygen, ASTM F1249: water vapor)

E.P. 3.1.7.: EVA for containers and tubing

Extractable and leachable tests with standard solutions

Gamma irradiation sterilization validation

These tests are conducted under standard settings with standard solutions. The data of these tests are available from the manufacturer.  

The authors discuss current and future disposable technologies and outline the validation and qualification steps that would be required for a possible disposable process stream.

Disposable Components in Aseptic Processing

Where possible, sterility of medicinal products is assured by heat sterilization. However, this cannot be used with every pharmaceutical product as it can alter or degrade the drug — particularly biologicals. In other instances, the gram-negative microbial bioburden level within the product is too high, meaning that heat sterilization would cause an undesirable concentration of endotoxin. 

Microporous membrane filters were invented in 1918 and came to use in the early 1930s.

  These filters either created a sterile effluent or a bioburden level reduction to a point of no concern in regard to any potential endotoxin contamination. Membrane filters in those days could not be standardized or reliably compared in consideration of retention performances. Instead, filter manufacturers performed microbial tests under different challenge conditions, and with organisms grown under diverse conditions and with different culture media. The lack of a standard made it impossible for the filter user and regulatory authorities to determine which filter should be designated the sterilizing grade filter.  

True comparability and performance analysis could only be performed when a standard microbial challenge test was defined that detailed: 					

The first steps towards establishing a standard were made with a document from the Health Industry Manufacturers Association (HIMA),

  which was later developed into ASTM standard F 838-83 and is now ASTM standard F 838-05.

  The focus on this standard for microbial testing led the industry to optimize sterile drug processing using filtration sterilization. The present biopharmaceutical interventions of fermentation-based drugs in medical treatments are the most recent consequences of ASTM standard F 838-05. 

A quarter of a century later, the standard still serves as the bedrock of organism quantification by which the efficacy of drug sterilization by filtration is determined. 

The first sterilizing grade filters were rated 0.45 μ and were used to remove yeast, mould and bacteria from beverages and pharmaceutical fluids. The definition of pore size rating commonly used is not derived from direct measurements, but from the rates of flow of fluids, air or water, through the filters. In filtration practices, however, emphasis is so heavily on particulate retention that it is sometimes popularly, but erroneously, believed that filters are classified according to the actual physical dimensions of their pores.  

Much ingenuity has been exercised in developing methods for measuring the size of filter pores. Flow measurements of both air and water have been widely used and resulted in an ASTM standard procedure (Figure 1).

  Other tests including mercury and/or air intrusion under high pressure have been employed and,

 more recently, vapour pressure analysis.

  New mathematical insights have also been brought to bear.

A first step towards a challenge test definition was occasioned by an incident of bacterial filter penetration. In 1967, Bowman 

. published the discovery of a smaller, water-born organism,

  which penetrated the 0.45 μ-rated filters that were then considered the sterilizing grade membranes. This finding resulted in the development and universal use of 0.2 (0.22) μ-rated membrane filters that could retain the smaller organism. The organism was identified as 

As with 0.45 μ-rated filters, the 0.2 μ rating was not standardized and multiple different challenge tests were again published by filter manufacturers. 

Sterilizing grade filters and their pore size ratings became critical tools within the industry and were, therefore, required to be tested accordingly. Any microbial breakthrough caused by an ill-defined rating could be detrimental to patient safety. Such occurrences were guarded against by assays enabled by the ASTM standard. 

The industry took action against the lack of a standard challenge test for sterilizing grade filters by forming, in combination with the filter manufacturers, HIMA. It established a standard microbial challenge test for sterilizing grade filters rated at 0.2 (0.22) μ. This was the first time the challenge organism, challenge level, growth media and filtration conditions, as well as challenge conditions, were defined. Another beneficial outcome of the efforts of this group was the term 'log reduction value' (LRV) as a description for the microbial removal efficiency of a filter. A filter with an LRV of seven is capable of reducing the organisms in the feed stream by seven orders of magnitude.

From that point onwards, the HIMA test methodology was used by filter manufacturers as the challenge test for 0.2 (0.22) μ-rated sterilizing grade filters, and as a verification method attesting that the filter will retain the challenge organisms at a level of 10

  of filtration area. Nevertheless, it only became an official standard when ASTM used its committees and membership to refine and convert the HIMA standard into ASTM standard F 838-83 in 1983, creating a solid, scientific benchmark. This standard was reviewed in 2005 with the consequent reissue of the standard as ASTM F 838-05.  

Integrity testing of filters is one of a series of interdependent activities for the preparation of sterile drugs. The key requirement of an integrity test is to establish a quantitative correlation between a property or characteristic of the filter of interest and its organism retention capabilities. This would, for example, address the needs of sterilizing filtration practitioners to identify filters of a particular pore size rating that have the necessary bacterial retention capability. 

FDA has defined a sterilizing filter as one that retains the classic challenge of at least 1×10

  of effective filtration area at pressures up to 30 psi (2 bar). This is best assessed by direct confrontation of the membrane by a proper organism challenge. However, once the filter has been tested it is contaminated by the organisms and, therefore, may not be employed as a process filter. Therefore, a surrogate test is used that is nondestructive to filters, and is based on correlation of pore size with organism retention. 

The recognition of a pertinent correlation involves more than the parallelling of two events; the relationship of the two actions that are seemingly correlated must accord to a technical logic as being the resultants of a common origin. The classical integrity test value or number correlates a filter's pore structure to its ability to completely retain challenges of a stipulated number of organisms per filter area. The significance of the integrity test is that it relates to its performance function. 

In the present practice, the membrane's largest pore size is probed by the bubble point test. Its value can be correlated with the membrane's ability to sustain the 1×10

 confrontation made by the use of the standard ASTM F 838-05 microbiological challenge procedure. On this basis, such membranes are experimentally qualified to be of the 'sterilizing grade,' and are, thus, labelled as being of the 0.2 (0.22) μ-rated pore size. They may then be selected for trials in actual product filtration sterilizations to ascertain their suitability to perform in given filtration contexts. 

Filter manufacturers have measured and correlated bubble point/diffusive flow values and LRVs for commercially available membranes. It is this correlation that serves as a nondestructive test for determining a given filter's organism retention capabilities. It is ASTM standard F 838-05 that makes possible the industry wide definition of LRV that is essential to this correlation. This is the information required to ensure the proper choice of membrane for use in filtrative sterilizations. 

Because of ASTM standard F 838-05, filtration with 0.2 (0.22) μ-rated filters became dependable instead of a gamble as to whether the so-rated filter was what it claimed. The FDA Aseptic Guidance document of 1987 was the first to define a sterilizing grade filter based on the ASTM standard description.

  The ASTM standard has also been adopted by many different agencies; for example, ISO's 13408-2 and PDA Technical Report 26.

This ASTM standard correlates the nondestructive integrity tests with the 

 retention characteristics of specific pore sizes. It is employed in defining the test limits of sterilizing grade filters. FDA recommends all sterilizing grade membrane filters to be integrity tested before and after utilization. As the filter user is unable to directly challenge every single filter used with organisms, filter manufacturers developed nondestructive integrity test methods, such as bubble point or diffusive flow, to serve as surrogates. However, these integrity tests must be correlated to the ASTM F 838-05 organism challenge test to determine their compliance with maximum allowable diffusive flow or minimum allowable bubble point limits (Figure 2). Such correlation tests are performed by filter manufacturers within their development laboratories and are published in validation or instruction documents. 

Furthermore, the standard became a cornerstone for the development of new filters in the 0.2 (0.22) μ-rated product area. Every time filtration products are improved or redesigned, these filters undergo standard bacteria challenge tests to verify the desired pore size rating. Moreover, while the ASTM F 838-05 standard defines the challenge test for 0.2 (0.22) μ-rated membrane filters, its guidance is of such value for filter manufacturers and users that its challenge conditions are also employed for other pore size ratings, except with different challenge organisms; for example, 0.45 μ-rated filters are challenged under ASTM test conditions with 

 being used as the challenge organism. For 0.1 μ-rated filters the commonly used organism is 

. New standards for challenge tests of different pore sizes could, potentially, be based on ASTM F 838-05, as the test method is already 'unofficially' used by many filter manufacturers to verify pore size ratings. 

In 1995, FDA employed the ASTM standard to further enhance sterility assurance by filtration. Most challenge tests performed before that point were commonly carried out on a water or broth basis under the teaching of FDA's challenge standard. However, the agency required verification of the sterile filtration performance of the filter under actual process conditions governing the filter's use and, if possible, with the actual drug product being the challenge fluid. The challenge level and the organism type stayed the same, and the challenge conditions and fluid stream utilized were adapted to production process reality. Again, this requirement has been prescribed by multiple organizations and guidances. A specific decision matrix has been established to define which actions should be taken when the challenge fluid is incompatible with the challenge organism (Figure 3). 

So successful were the 0.2 (0.22) μ-rated membranes in producing sterile effluent that the identification of a filter's pore size rating by way of integrity testing was taken to ensure that its use would yield a sterile effluent. The guiding assumption, however, was dependent upon sieve retention or size exclusion being the organism retention mechanism and was based on 

With time, it was found that the 0.2 (0.22) μ-rated membranes did not necessarily result in a sterile effluent. It would seem that the key correlation based on the ASTM developed standard was not acceptable.

Further investigation showed that the organisms that escaped capture by the 'sterilizing' 0.2 (0.22) μ-rated membranes had undergone size diminution following immersion in liquid vehicles of limited nutritional value. Far from proving inadequate, the correlation demonstrated its applicability for retaining smaller organisms by the use of membranes of lower pore size ratings, namely, the so-called 0.1 μ-rated filters. 

Filtration practitioners' understanding that the sizes of organisms were capable of undergoing change was heightened by Leahy and Sullivan who found that the size of the 

 microbes depended upon the method of cultivation.

  This emphasized the need to standardize the procedure whereby the very organisms used to confront the filter pores would repeatedly and dependably be cultivated to match in size. The operational protocol set forth in ASTM standard F 838-05 made this possible, eliminating the issue of organism size variability. The standard satisfied the need for organisms of sufficiently similar size to serve as standard size particles. Consequently, they were suitable for the assaying of their retention by pores of different sizes. This legitimized comparisons among competitive filters on technical grounds as the standard eliminated challenge test variability. 

To increase effectivity of the sterilizing filter, the substitution of the 0.1 for the 0.2 (0.22) μ-rated membrane was suggested. However, of the seven 0.1 μ-rated filters tested, only four were found efficacious. The more generalized use of 0.1 μ-rated filters was, thus, unwarranted.  

Correlation of LRVs with the assaying of organism counts could not be applied to a pore size classification that lacked industry identification. A suitable test organism whereby the ASTM standard F 803-05 test could provide the necessary correlation had first to be agreed upon. 

In the circumstances, the use of the tighter filters was limited by practical considerations such as lower rates of flow. The substitution of 0.1 for the 0.2 (0.22) μ-rated filters is recommended where organism shrinkage is possible — especially for filtrations of long duration. The use of the tighter membrane awaits its LRV correlation to the native bioburden organism colony counts of its effluents. The native microbe appropriate to the inquiry requires identification. The ASTM standard creates a basis to perform such investigations. 

The success of filtration sterilization applications encouraged competition between filter manufacturers that some believe bordered on marketing rather than technical considerations. 

In the circumstances, the fact that ASTM standard F 838-05 existed empowered the pharmaceutical industry to secure scientific-based correlation data that muted commercial concerns. Beneficially, the ASTM standard devoted to technical matters, in quieting commercial considerations, attained a laudable goal. Society and the patient were the beneficiaries of an ASTM standard-setting effort. 

Bioburden studies, integrity testing and process validation are the building blocks of filtration sterilization exercises. The contributions of ASTM standard F 838-05 to bioburden studies and to filter integrity testing have been discussed, along with its influence on fulfiling validation requirements. It created a well-defined testing range and activity flow for the validation efforts essential for verification of the filtrative sterilization operation. Without this industry standardization on behalf of a specific and acceptable test range, individual efforts would be confined to limited goals. The basis for the enhanced challenge definitions, and for the adoption of a true process validation approach was ASTM F 838-83 (now 838-05).

Although realized in 1983, ASTM's standrad test method for determining bacterial retention of membrane filters used for liquid filtration is still hugely beneficial to today's pharmaceutical industry.

Assuring sterility with ASTM F 838-83

Book reviewers come in all stripes. They are generally perceived to be experts in the discipline that is the subject of the book. Their extensive knowledge of the subject permits them to judge the correctness and depth of the information being imparted by the author. 

 Industrial Bioseparations: Principles and Practice by Daniel Forciniti, Wiley-Blackwell, Oxford, UK, 2008, 309 pp., ISBN: 978-0813820859 

Daniel Forciniti conveys a great deal of information in an impressive work called 

Given the breadth of this burgeoning field, few are qualified to review this book. 

But the novice (such as myself) may perhaps also serve as a reviewer. He or she can attest to the author's helpful guidance, to the principles that form the structure of the book, and to the aptness of the tools and practices the author uses to teach the reader about the subject. In this sense, the reviewer's critique reflects his or her experience reading the book. 

In the book's preface, Forciniti refers to the wealth of biotechnological advances that now produce therapeutic drugs and other compounds. Because the end products are often proteins made from the genetically altered cells of organisms such as bacteria, plants, and animals, they must be isolated from the fermentation mixes that produce them. The techniques required for this purpose derive from biological operations that require engineering skills based on the physical sciences. These skills have seldom, if ever, been dedicated to the resolution of such complexities. It should be emphasized that more than an elementary knowledge of both the biological and physical sciences is required for this isolation, in a combination that translates into an engineering application. 

Forciniti undertakes and achieves his goal of teaching the principles and activities involved in bioseparations engineering practices. He teaches in the definitive language of mathematics, and his approach to technical causes and effects will oblige the purists who require certainties. Those secure in conclusions based on thermodynamics will applaud Forciniti's mathematical logic. His language is impressively clear and exceptionally instructive and explanatory. 

The book discusses the production and purification of biomacromolecular entities. It thoroughly explains solid– solid, solid–liquid, and liquid–liquid separations. The principles that define the various states of matter are made plain in the book. The requisite techniques for isolating and purifying the diverse products are considered and evaluated in abundant detail. The techniques of flocculation, centrifugation, filtration, chromatography, and precipitation are investigated. Discussions of cell disruptions, protein molecular structures, freeze-drying, osmosis, and reverse osmosis are not neglected. 

The book also offers practical information that extends to pertinent materials and methods, even to pumps, valves, and mixers as well as to separation cascades and types of chromatography and centrifugation. The breadth of Forciniti's coverage of technical topics is awesome. 

In dealing with solid–liquid separations, Forciniti discusses the one discipline with which the reviewer has some technical familiarity (namely, filtration). The book discusses the materials and methods of conventional industrial filtrations dealing with the plate-and-frame system and rotarydrum, vacuum-activated equipment. Forciniti also discusses unconventional filtrations such as ultrafiltration and microfiltration in both the dead-end and cross-flow modes. He explains Darcy's Law both mathematically and verbally in satisfying detail. 

Forciniti is obviously familiar with the finer filtrations based on membranes in large-scale pharmaceutical manufacturing and in making semiconductor chips. He cites appropriate sources and literature on the subject of filter manufacturing. 

The book, necessarily limited at 309 pages, provides a surprising amount of operational details and imparts valuable information to the reader. As Forciniti made evident in the preface to his book, he did not wish to design an encyclopedic work, but rather a well-balanced book. He attained his goal with considerable distinction. This volume will serve as an invaluable introduction and guide to those embarking on careers in the discipline of bioseparations. Foriciniti deserves our thanks for promoting knowledge of this important endeavor. 

 is a principal at Capitola Consultancy, 8103 Hampden Ln.,Bethesda, MD 20814-1244, tel. 301.986.8640, 

A well-balanced guide to industrial bioseparations provides valuable information.

Bringing Certainties to a Burgeoning Field

A septically produced liquid pharmaceutical and biopharmaceutical products are usually sterilized by filtration. The filtration process must be validated to ensure that it is capable of removing all microorganisms from the product. Validation consists of challenging the filter with a suspension of 

 and analyzing the filtrate for microorganisms. The filtrate must be sterile. 

Filters used in production should be equivalent to the filters used in the bacterial-challenge validation studies. Because actual production filters cannot undergo bacterial-challenge testing, integrity testing is performed to demonstrate bacterial-retention equivalence. If the integrity-test values obtained for the production filters are equivalent to those obtained for the filters successfully passing bacterial-challenge testing, then it is assumed that the filters have the same bacterial-retention properties and that the filtered pharmaceutical product is, therefore, sterile. 

Integrity testing for the hydrophilic filters used in pharmaceutical production relies on the measurement of gas flow through wetted membranes. This flow can be classified as diffusive or bulk and is sometimes a combination of both. Fick's Law of Diffusion shows that diffusion of the test gas through the liquid-filled pores in the membrane is a function of the diffusion constant and the solubility of the test gas in the liquid at the test temperature, the pressure differential of the test gas across the membrane, the thickness of the liquid layer, and the area and porosity of the membrane (1). Diffusion is not directly related to pore size although, as will be shown later, there is an indirect correlation. Bulk flow occurs when the test gas flows through the nonwetted or empty pores of the membrane. Open pores occur because the filter membrane has been incompletely wetted or because the bubble point of the membrane has been exceeded. Bulk flow primarily is a function of the size and number of the open pores, the thickness of the membrane, and the pressure differential of the test gas across the membrane at the test temperature. 

As indicated previously, gas flow through wetted membranes is diffusive, bulk, or a combination of both. Figure 1 shows the relationship between gas flow and differential pressure for a typical membrane filter. Similarly, because it is an arithmetic function, pure bulk flow is a straight line whose slope is determined by the size and number of open pores, assuming all other variables are held constant.  

The knee area of the curve is where the influence of the bubble point is manifested. Here, the largest pores of the filter become unblocked as the applied pressure overcomes the capillary forces within those pores, and bulk flow begins to increase the slope of the curve. It is also in this region that diffusive flow begins to increase because the thickness of the liquid trapped in the largest pores begins to decrease as a result of the increasing pressure differential. Therefore, within the knee area of the curve a complex relationship exists between diffusive and bulk flow, both influenced by the pore structure and pore-size distribution. 

Validating the bacterial-retention capabilities of sterilizing-grade filters involves challenging the filters with a suspension of 

 (ATCC 19146) at a level of at least 1 × 10

  of filter area, resulting in a sterile filtrate. Specific details and options may be found in "PDA Technical Report No. 26

(2). The integrity-test values of the filters used in the bacterial challenge studies must be known to form the basis for ensuring the filters used in production have equivalent bacterial-retention capabilities. In addition to the integrity-test values, the manufacturing processes and quality-assurance systems of the filter manufacturer must be adequate to ensure consistency of the filters within and between each manufactured lot with respect to nominal pore size, pore-size distribution, and membrane thickness and area.  

Bacterial retention is assessed by challenging filters of the intended type having a range of integrity-test values and analyzing the bacterial-passage results. Ideally, the retention pattern observed will allow identification of physical integrity-test values above or below which, depending on the type of integrity test, there is virtually no probability of bacterial passage. For example, with a series of filters having a range of bubble points and equivalently challenged, there would be a value below which bacterial passage always occurred, a range where passage sometimes occurred, and a value above which passage never occurred.  

It is important to identify the integrity values above and below which bacterial retention and bacterial passage can be expected. This ensures that the correlation between the integrity-test value and bacterial retention is valid and there are not other factors such as pore-size distribution and anisotropy that are potentially influencing the perceived correlation. 

During development of what he referred to as the "forward flow" test in 1973, Dr. Pall performed bacterial-challenge testing on a series of 43 cartridges having water-wet forward air flow values ranging from 16 to 900 cm

/h at a pressure differential of 10 in of mercury (4.9 psi) (3). The results of this testing showed that no filter with a flow of less than 199 cm

/h allowed bacterial passage. On the basis of those results, he established a maximum flow limit of 100 cm

/h for an acceptable production filter element, building in a safety factor. However, the testing also showed that there were filters with forward-flow values of 210, 258, 450, and 645 cm

/h that retained the challenge organism and filters with forward-flow values of 199, 238, 471, and 600 cm

/h that allowed bacterial passage. The study did not establish a point above which bacterial passage always occurred; therefore the correlation between the integrity-test value and bacterial retention was not fully elucidated.  

Because the forward-flow test as described relies on diffusive flow, which in terms of the filter membrane is a function of porosity, area, and thickness and is not directly related to pore size, the bacterial-retention results obtained for the higher forward-flow values could be indicative of pore-size distribution differences (or possibly thickness variations) among the tested membranes. Had these tests been carried out at pressures closer to the bubble point as is done today, differences in pore-size distribution might have been revealed as a result of the pressure-induced thinning of the liquid layer in the larger pores and better correlation obtained with bacterial retention at the higher diffusive-flow values. Nonetheless, Pall's efforts served as the basis for developing the automated filter-integrity tests in use today. These tests operate at considerably higher pressures, capturing the influence of the knee area and providing effective correlation between bacterial retention and the integrity-test value. 

It is important to note that the integrity-test values were obtained on clean filters, before commencing the bacterial-retention tests, eliminating the influences of filter loading on filter porosity and diffusive-flow values. 

Another validation consideration is to test the filters after sterilization and bacterial-challenge testing to evaluate the influences of filter loading and the sterilization process on the integrity-test values. 

The sterilizing filtration process is designed to remove viable and nonviable particles from the fluid passing through the filter. The particles removed from the fluid either remain on the surface of the filter membrane or are trapped within the membrane matrix. 

It has been shown that hydrodynamic flow is preferentially directed to the larger pores in a filter membrane (4). This means that hydrodynamic flow directs the suspended particles to the largest pores. However, because the largest pores are far less numerous than the average-sized pores, particles initially tend to be captured by the many smaller pores. As the smaller pores become blocked, the hydrodynamic flow directs more of the particles to the larger pores. The larger pores now may become blocked or partially blocked because of bridging, which is promoted by high levels of particulates competing for the pore openings. This phenomenon is corroborated by Grant and Zahka (5). They showed that during the filtration of a dilute suspension of silica particles there was complete retention at the beginning of the filtration, followed by particle passage. The retention then increased as the filtration continued. Similar results were obtained by Roberts and Velazquez while filtering latex particles (6). Thomas 

 found that the successive changes affecting a filter's pore-size distribution as a filtration of 

progressed showed the larger pores to be less immediately engaged in the retention of particles (7). 

Effect of filter loading on integrity-test results

The effects of filter loading on diffusive flow and the bubble point have been evaluated; however, comprehensive studies remain to be performed, and understanding of these effects is currently incomplete. Trotter 

 showed that the diffusive-flow values at 45 psi began to change at a bacterial cell loading of 1 × 10

, while the bubble point remained essentially unchanged until the bacterial cell loading approached 1 x 10

Particle capture inherently reduces the porosity of the filter, thus decreasing the diffusive flow, and it increases the bubble point as the largest pores become blocked. Also, the effective thickness of the membrane increases as material accretes on its surface. These effects have implications with respect to the integrity-test values and their correlation to bacterial retention. Certainly, porosity reduction and the increase in thickness depend on the feed stream and the properties of the particles (gel or solid) removed from it. 

Filter loading influences diffusive-flow and bubble-point values, although not to the same degree and not in the same sequence according to published studies. Postfiltration integrity testing evaluates filters that have been subject to an unknown level of particulate loading; therefore the influence of the particulate loading on the integrity-test results is also unknown. It is reasonable to assume, however, that as particles are arrested by the membrane, the porosity of the membrane decreases and the bubble point increases. This assumption has been confirmed experimentally (8). Testing under different conditions or with different membrane types and configurations could produce different results, however. 

Extensive validation testing using multiple lots of product representative of normal production is necessary to determine the potential effects of filter loading on the postfiltration integrity-test values. In any case, the precise effect of the filter loading on the postfiltration integrity test cannot be known with certainty; therefore, it is not possible to ensure that filter loading has not masked a defect that initially may have been present in the filter. 

Regulatory agencies differ in their positions regarding the need for prefiltration integrity testing of membrane filters used to sterilize liquid pharmaceutical and biopharmaceutical products. Annex 1 of the 

 specifies integrity testing of the sterilized filter before and after use. FDA's 2004 

Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing

 suggests that integrity testing can be performed before processing and that integrity testing should be routinely performed post-use to detect leaks or defects that may have occurred during the filtration process. The FDA guidance does not take into account potential changes in the validated prefiltration integrity-test values as a result of filter loading. 

Because the validation studies correlating bacterial retention with integrity-test results use prefiltration integrity-test values, albeit with a safety factor, the only reliable way to ensure that the filters used in production are equivalent to those used to establish the integrity-test specification is to perform a prefiltration integrity test. The postfiltration integrity test then serves its intended function: to ensure the filter has not been damaged during the sterilization or filtration process.  

 is president of the Williamsburg Group, LLC, 18907 Lindenhouse Rd., Gaithersburg, MD 20879, tel. 301.938.4266, 

 is group vice-president of global product management, bioprocess of Sartorius Biotech Inc. 

 is a consultant for Capitola Consulting Company. Madsen and Meltzer are also members of 

*To whom all correspondence should be addressed. 

Keywords: filtration, integrity testing. sterilization 

 1. F. Hofmann,"Integrity Testing of Microfiltration Membranes." 

 "PDA Technical Report No. 26: Sterilizing Filtration of Liquids," 

 3. D.B. Pall, "Quality Control of Absolute Bacteria Removal Filters," 

, "Integrity Test Measurements—The Effects of Bacterial Cell Loading," 

 5. D.C. Grant. and J. G. Zahka, "Sieving Capture of Particles by Microporous Membrane Filters from Clean Liquids," 

 6. K.L. Roberts and D.J. Velazquez, "Characterizing the Rating and Performance of Membrane Filters for Liqid Applications Using Latex Spheres," 

 during Microbiological Validation of Filters." 

 "Investigation of a Filter Structure by Microbial Retention Studies: A Synthesis and Elaboration of Prior Findings," 

Pre-use integrity testing of sterilizing-grade filters eliminates the potential adverse effects of filter loading on the integrity-test results, allowing unambiguous correlation with the integrity-test specification established during filter-validation studies.

The Importance of Pre-Use Integrity Testing in Sterilizing Filtration

Microporous membrane filtration is the technique often applied to the aseptic processing of drug preparations. This is especially appropriate when the ingredients are heat labile. Certain filter performance qualities are of specific interest in such filtrations, namely, the extent of organism removal, the rate of liquid flow, and the total throughput volume. Essentially, it is the numbers and sizes of the pores relative to the number and sizes of suspended particles that determine the filter retention performance, although there are other factors that also affect organism removals (1). The total aggregate space of the pores within the solid filter matrix represents the membrane's porosity. It is constituted of the total number of pores of whatever dimensions. The importance of pore size is obvious. The filter pores are the sites through which the liquid flows. Simultaneously, its suspended particles, such as organisms, are retained at the pores. Ultimately, the throughput is limited by the accumulation of the particles at the pores causing their blockage.  

Microporous membranes prepared by the reverse-phase casting process are available from several manufacturers in a series of pore sizes ranging from 0.04 to 8 μm. Curiously, the rating values are not determined by direct pore-size measurements, although several different methods for sizing have been proposed.  

Tests such as bubble-point measurements and bacteria challenges are used to assign pore-size ratings. The propriety of translating bubble-point values into pore-size ratings will be discussed later in this article. The assigned numbers are meant to imply particles-size retentions, not dimensional mensurations from which flow properties might also be derived. The bubble points are indicators of the largest size pores present in a membrane; the focus in filtrations being chiefly upon particle retentions. 

The significance of pore sizes lies in their implication to particle-size retentions. The quantitative characterization of pore sizes derives from bubble-point measurements coupled with bacterial challenges. These tests are correlative. The former does not compromise subsequent use of the filter. It is a nondestructive test. The latter, although more direct in its diagnosis, is a destructive test in that it contaminates the filter with the test organisms; thus obviating its later application to process filtration. 

The organisms used in the microbial challenges differ in accordance with the filter's presumed pore size. The log reduction values necessitated by FDA's definition of a sterilizing filter is the retention of  1 × 10

  colony forming units (cfu) per square centimeter of effective filter area (EFA) (2). Such a retention produced against a 

 ATCC-19146 confrontation characterizes the 0.2/0.22 μm-rated membranes. 

 is the organism commonly used in challenging 0.45 μm-rated membranes. Because Bowman 

 determined that the 0.45 μm-rated membranes retain 

  level, that test is sometimes used for testing the 0.45-μm membranes (3). The 0.1 μm-rated pore size is mostly tested against 

 It is fair to say that the testing of  0.1 μm-rated membranes is not yet fully in place within the industry. 

Intermittent bubble-point measurement also is used as a process guide during the membrane-casting operation to ensure that the intended pore-size ratings result. The point being made is that the relevant measurements being performed are of bubble points, not of pore sizes. From these, as will be discussed, pore-size ratings and log reduction values (LRVs) are derived. 

The pore-size ratings are the remnants of early efforts at membrane classifications that developments in pharmaceutical filtrations have reduced to rather insignificant utility. Pore-size ratings are administrative devices. They serve only as parts numbers or catalogue listings for filter manufacturers. The selection of filters for sterilizing applications is made primarily on the basis of the LRVs that the various filter types impose upon 

 challenges in accord with FDA's definition of a sterilizing filter (2). The relationship of LRVs to experimentally determined bubble-point values enables use of the latter to identify potential sterilizing membranes subject to verification by microbiological assays performed by the filter manufacturer. The putative pore sizes are affixed accordingly. The membranes that meet FDA's stipulation of retaining the 1 ×10

  challenge are listed as being rated as 0.2/0.22 μm. 

Microporous membranes can be prepared by various methods. Among these is the track-etch process wherein a polymeric, dielectric film is bombarded with heavy-mass fission fragments, followed by an alkali etch of the radiation-damaged pathway. This process creates pores that are straight through and regularly cylindrical in shape. Their diameters can be accurately measured with scanning electron microscopy. Despite the relative regularity of their pore structures, these membranes do not usually find application in pharmaceutical processing. The membranes used in pharmaceutical processes are almost exclusively prepared by the phase-inversion technique, generally referred to as the "casting method" (4). 

Little is known about the numbers, sizes, and shapes of the pores of microporous membrane so prepared. The membrane structure usually is pictured as being analogous to that of a polymeric sponge. A hypothesized oversimplification of the pore passageways is that of irregular and tortuous capillaries that are, therefore, more extended in length than the filter's surface-to-surface thickness. The pores are marked by irregularly restricted diameters that provide the choke-points that interfere with particle passage. However complex, the pores are pictured as being essentially cylindrical and composed of interconnected spaces extending through the depth of the polymer matrix.  

The very concept of a definable "pore" is an artificiality when applied to microporous membranes other than the straight-through columnar pores that characterize the track-etched variety. The complex geometry of the sponge-like membrane results in the pores having ratios of cross-sectional areas to perimeters, called the "hydraulic parameters." These vary over the entire thickness of the membrane (5). A membrane's depth can be constructed of several superimposed unit planes that in their aggregate impose their effect on retention and flow rates (6). The "pores" so considered are presumably connected throughout the unit planes to constitute pathways for fluid flows. However, where particle retentions interfere flow redistributions may result through new "pore" alignments. The "pore" concept arises as a hypothetical construct useful in understanding filter performance. Unlike the track-etched pores, they are not integral, structural pathways for fluid flow.  

The pore structure derives from a cast polymer solution wherein the polymer chain segments are separated from one another by distances that reflect the degree of dilution. It is the inter-segmental distances among the polymeric chains that in their interconnections prefigure the "pores" of the finished membrane. Formulae of various polymer concentrations give rise to different intersegmental separations, ultimately to different porosities, when by proper manipulations the polymer is precipitated as a gel, to be washed and dried to its solid, microporous membrane state. There is inevitably a pore-size distribution and some anisotropic pore shape formation (4). 

 Figure 1: Free-floating soap bubbles.			

It is hypothesized that the formation of the microporous membrane structure accords with the known phenomenon of "soap bubble clustering" (7). The reasons for this resemblance is that in both cases there is the coming together of spheres whose spatial clustering is under the influence of area-minimizing forces. The geometric consequences of these forces is known from the study of soap bubbles (8). Polygonal facets characterize the resulting spaces of a free-floating cluster of soap bubbles (see Figure 1). In the pore formation, the nonsolvent of the casting solution takes the place of the air of the bubbles. In support of such structures, Figure 2 is that of a detergent foam confined between glass plates. The polyhedral spatial structures are obvious. Figure 3 is of a reticulated polyurethane (polymeric) foam. The cellular pores can be seen, in fact, to be polygonal in shape. The phenomenon of clustering through polyhedral spatial arrangements is manifested in other settings as well. It is a trait of zeolitic molecular sieves whose interconnection is through the open panels common to contiguous polyhedra, albeit caused by crystal-packing rather than area-minimizing forces (7). There is, therefore, technical support for the concept of polyhedral microporous structures. 

Figure 2: Detergent foam between two glass plates.			

In the case of the microporous membranes, the pores, as stated, are formed from the open intersegmental areas prefigured in the casting solution. They are hypothesized to be of various polygonal shapes, framed by polymeric struts and walls, and to be, like the zeolites, interconnected by openings in their common walls. It is these openings that are seen to be the metering and retaining pores of the membranes. As stated, casting solutions of various polymer concentrations give rise to separate but rather similar intersegmental distances. It is these spaces that are ultimately transformed into the membrane pores that, in consequence, are also similar in their dimensions—except for the pore-size distribution that results from the casting solutions' deviation from an ideal homogeneity. 

Figure 3: Reticulated polyurethane foam.			

Filtration is not a simple sieving process, except perhaps in the case where the filter is a track-etched membrane with pores passing straight through a much thinner film (~10 μm). As stated however, for the membranes prepared by the casting process (thinness ~150 μm), a fluid on passing through encounters pores of different diameters and surface areas. Particles are separated from the fluid by adsorptive attachments to the pore surfaces as well as by the size exclusion mechanism. Thus, the meaning of the average pore size as reflecting the size of a restrictive diameter within a pore passageway is necessarily an oversimplification. Nevertheless, the events taking place at the pores are depicted as if the pores were continuous and integral paths through the depth of the membrane. 

Membrane characteristics are assessed because of their pertinence to aseptic processing. However, the pore-size distribution, despite being an important structural feature that influences both flow and retention, is not among them. It is seldom known or investigated although an ASTM method  based on airflow rates enables its assay (9). The reason for not assaying the pore-size distributions of filters is that complete organism removal is dependent upon the largest pores of the pore-size distribution retaining the smallest particle of the particle-size distribution. This is the singular circumstance wherein an absolute filtration can eventuate. Given this felicitous situation, only the size of the largest pore has pertinence. In fact, however, the distributions of neither the pore nor organism sizes are likely to be known by the filtration practitioner. Depending on the relative numbers of pores and particles and their sizes, particles may encounter the larger pores of the distribution to escape removal. These larger pores of a distribution are assayed by bubble-point measurement recorded in pressure units (psi or bar) rather than in dimensional units (μm). The accompanying smaller size pores are ordinarily not quantified or measured because they are not seen to influence retentions.  

 Numerous methods have been used to assess pore size (10). Early on, mercury porosimetry was the method of choice (11–13). In this procedure, mercury is forced under pressure to penetrate into membrane pores. This process is performed at increasing differential pressures, Δ

, the smaller the pores that the fluid metal can intrude. Quantitation of the different pore sizes relates to the volume of mercury that is intruded in filling the pores at each of the progressively increasing differential-pressure stages. Airflow porosimetry studies also have been performed (14, 15).  

The porosimetry method is flawed by the assumptions necessary to its application. It is not suited to polymeric materials that are at temperatures above their glass-transition points and whose pores are, therefore, liable to stretching and distortion under the pressures of the intruding fluid. Also, the averaging of volume changes required by the technique may mask the true dimensions of the "pores." 

Pore sizing based on the retention of organisms of presumably known sizes has been widely investigated (14, 16–18). Pore sizing attempts also were made using latex beads of very narrow particle-size distributions (19, 20) (see Table I). These latter methods, in conjunction with the use of surfactant, have the advantage of eliminating adsorptive effects from those ascribed to sieving (21) (see Table II). Various surfactants were found to differ in their influencing the sizing results (22). The assigned pore sizes assumed the particle to be spherical and the area of pore restriction to be circular in shape. Simplifying assumptions were necessitated to finesse the shape factor that is operative in retentions. The exclusivity of sieve retentions also was assumed, ignoring adsorptive arrests, and at best allowed inadequately for pore-size distributions. Reductions in organism sizes resulting from contact with given liquid preparations, shown to be possible by Sundaram 

 (23) were not investigated. Nor have the possible plasticizing effects of the suspending liquids on membrane pore-size alterations reported by Lukaszewicz and Meltzer in 1980 been investigated (24). 

Table I: Percent retention of various size latex particles for 0.2 Î¼m-rated membranes.			

The results of these trials usually were judged to be of limited value. This is typical of efforts where simplifying assumptions are used to support a hypothesis, and the very conclusions are limited by the inherent arbitrariness of the necessary premises. They are complicated by the fact that the definitions of the particles themselves may depend on the particle-measuring methodologies, on the procedural protocols, and on the measuring devices used (16). 

Pore-size ratings are so unrelated to actual dimensions and so subject to anomalous interpretations as to make substantial dependency upon their values an unwise choice. Moreover, the means of measuring them are questionable. The pore-size rating system at best provides a qualitative differentiation.