Kevin Jones, Whatman Inc. (Florham Park, NJ)
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A track etched membrane can provide accurate filtration. In this photo, 2.3 micron latex beads are captured on a 2.1 micron pore size membrane
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Membranes and filters have played an important role in IVD technology since the earliest days. Although they have often been dismissed as just a component of the assay with a behind-the-scenes function, these separation materials have been assuming increasing importance in IVDs. Regardless of how their role is perceived, the recent advances that have been seen in IVD technologies and applications have been paralleled by advances in membrane and filter technologies.
Today’s IVDs are far superior to their earlier counterparts in fulfilling the demands of a growing marketplace. IVDs have rapidly progressed to the point where it is now possible for technical and nontechnical users alike to conduct accurate and reproducible tests in adverse conditions. Once merely a concept to fire the imagination, IVDs that operate at the molecular level now enable genetic abnormalities to be identified. Membranes and filters, still fundamental to the manufacture of IVDs, however futuristic, have contributed directly and indirectly to the improvement of the lives of millions of people by the use of IVDs in healthcare.
IVDs and Separation Materials, Advancing Together
The debate as to whether advances in IVDs have led to improvements in membrane and filter technology or whether advances in membranes and filters have made possible IVD advances derives largely from differences in perspective. Those presenting the case for IVDs having primacy would argue that the increasingly demanding requirements of ever more sophisticated diagnostic assays forced membrane and filter manufacturers to respond. On the other side are people who would say that the advances made in membrane and filter technologies have allowed the IVD industry to develop new methods and applications that exploited the new capabilities of these materials.
The debate in many ways is academic. Whichever side one chooses, there is no denying that IVD devices and membranes and filters have a close relationship, that each technology contributes to and supports the other. Modern assays would be impossible without the developments lately seen in filtration and membrane properties in any case. A broad review of today’s technologies and applications will help to understand the relationship between membranes and filters and IVDs.
In essence, membranes and filters provide a means of performing a separation, either the removal of particles from a solution or the separation of blood components, or the exchange of salts in a solution. Many of the materials used in early filtration applications are primitive by today’s standards. The properties of those early membranes and filters were determined directly by the materials and methods used in their manufacture.
While it is true that many of these raw materials are still used today, advances in separation technology and in the manufacturing processes used to produce membranes and filters have eliminated the vast majority of their undesirable characteristics. Also, many new materials have replaced early ones. Whether woven, cast, or etched, today’s custom-engineered membrane and filter products retain their basic function but also exhibit predictable, designed-in properties and performance.
Filtration Media in the Background
Without the contributions made by modern filtration and membrane technology, IVDs might bear little resemblance to those with which we are familiar. It is difficult to imagine the recent success and advancement of the IVD industry and its products without device components such as monoclonal antibodies, recombinant proteins, and molecular probes. Membranes and filters play a vital role, somewhat surprisingly perhaps, in making each of these essential components available to manufacturers. The availability and, more important, the uniformity and reliability of media for sterile filtration have made production of these biological IVD components less cumbersome by effectively minimizing the threat of contamination. Without them, IVD manufacturers would face the possible loss of components due to biological or physical intrusions causing impurity. That a large number of companies are able to develop and produce the necessary antibodies, proteins, and molecular probes is attributable in part to the ready availability of prefabricated filtration units. Indeed, many reagents used in IVDs originated in small research and production laboratories that rely on membrane and filter products.
Allowing production of a biological component under sterile conditions is only the beginning of the contribution to IVD manufacture made by membranes and filters. With few exceptions, biological components that are used in IVDs require purification. From macrofiltration to microfiltration and on to ultrafiltration and diafiltration, the separation materials industry has a range of products to meet the needs that define each stage of purification. Starting materials must be clarified, buffers must be made particulate-free, fractions must be concentrated, buffers must be exchanged, and products must be sterilized. Filtration products are available for accomplishing each of these tasks efficiently, reproducibly, and reliably.
Dedicated or specialized products, from highly porous filters to ion diffusion membranes, have been devised for each application. However, it is often the case that one type of membrane or filter performs several different activities. Consider hollow-fiber units used for in vitro antibody production. These combine several levels of filtration capability. Effectively, these so-called “artificial mice” provide macrofiltration (for cell retention), ultrafiltration (for concentration of antibodies), and diafiltration (for diffusion of culture nutrients). Such devices may become increasingly prominent as pressure on the industry to move away from in vivo monoclonal antibody production mounts. Other devices that offer multiple filtration functions and play an important role in the preparation of IVD reagents include tangential-flow units, stirred cells, and centrifugal apparatus.
Separation Media on Center Stage
The examples just given illustrate the more or less hidden and nonintegral significance of membrane and filter technology in IVD development. Separation materials often assume a more prominent role in the IVD device itself, however. The contribution is indirect where the membrane or filter does not participate immediately in the result (the test reaction) and direct where it does. But whether acting directly or indirectly, the separation media are just as crucial to the performance of today’s IVDs as they are in the development of those same devices.
One of the most widespread indirect applications of filtration materials associated with IVDs is sample preparation. Samples used in IVDs have always required some amount of preprocessing, and this has become even more important with many of today’s sophisticated IVD techniques. As the minimum detectable level of analyte decreases, the susceptibility of the assay to interference increases. The presence of contaminants, especially in molecular diagnostics, can lead to erroneous results.
Membrane and filter technology can be used actively to target specific components for sample depletion or enrichment. A very common IVD sample purification application is the targeted separation of red blood cells and leukocytes from plasma. With the availability of new separation materials, device manufacturers can incorporate selective filtration capabilities into their IVDs, making possible the performance of onboard blood separation. The rapid increase in the number of whole blood assays available on the market serves as evidence of the efficiency and reliability of the filtration elements used.
Separation materials play another important active role in today’s IVDs. Many modern assays are performed on complex, automated instruments that, as with electronics generally, have become smaller. This miniaturization leads to a lower tolerance of particulate contaminants. Just as filters can be employed to remove blood cells or to clarify samples, so can they remove particles from the reagents used in these new platforms. Regardless of the type of test or the nature of the contamination, improperly preparing a sample or reagent can lead to instrument downtime. More important, a poorly prepared sample can cause the worst possible assay outcome: an inaccurate test that results in a misdiagnosis. State-of-the-art separation media enable IVD manufacturers and end-users to minimize undesirable and unacceptable risk factors for these possibilities.
Immunochromatographic Tests
From reagent production and purification to sample processing and instrument protection, the indirect role of membranes and filters in IVD technology is clear. But, as suggested earlier, separation media often play a direct part in IVD testing. One of the best examples of this more active function is their use in rapid IVDs, particularly the lateral-flow format frequently referred to as immunochromatographic tests (ICTs).
Early ICTs were based on strips of chromatography paper that had been passively sensitized with active components. With subsequent generations of the technology, covalent attachment of the reagents addressed some of the problems associated with their tendency to leach. Assay performance—most notably sensitivity and reproducibility—improved when the reagents were firmly immobilized to the support. (Activated-paper supports were used in other IVD formats as well, such as radio and enzyme immunoassays, and also commonly in allergy diagnosis.)
However, even with covalent immobilization of the reagents, chromatography paper was far from an ideal support material. This was due in part to the natural variation in any paper product. Also, the absorbency of papers meant that large sample volumes were required to run the tests. Further improvements were clearly needed if ICTs were to become as capable as the familiar products of today.
Nitrocellulose Membranes
The transformation of ICTs came with a change in the substrate. The binding properties of nitrocellulose membranes (NCMs) had long been known to those familiar with blotting techniques. When these membranes were introduced into ICTs, they very quickly replaced paper as the preferred support medium for the tests. Their higher protein-binding characteristics made activation of the support no longer necessary for achieving adequate reagent attachment. In addition, because NCMs were significantly thinner than their paper counterparts, smaller quantities of sample and components were sufficient for performing the test.
Thus it was that ICTs were first devised and then transformed by IVD developers using existing separation materials—first paper and then nitrocellulose—in applications for which they were not intended, adapting them as needed.
Tests that were more sensitive and required lower sample volumes fitted very nicely with the market requirements for ICTs. However, early NCMs had many undesirable characteristics. Variations in capillary rise time, thickness, binding properties, and surface characteristics all contributed to variation in ICT performance. Furthermore, the membranes were difficult to manipulate during the manufacturing process. As the use of immunochromatographic IVDs increased and new applications were developed, IVD manufacturers challenged makers of membranes and filters to provide them with materials that were better suited to the growing market.
Working in conjunction with the IVD industry, the membranes and filters sector strove to meet the needs of test developers. Media developers increased their knowledge of NCM production, improved the manufacturing processes for these membranes, and developed new membranes specifically for ICT applications. The newer NCMs were far superior to those originally used in ICTs. Rise times became more consistent and could also be tailored to meet specific needs. Gradually, faster-wicking materials that still yielded a high level of sensitivity were adopted as the norm. NCMs with a variety of different, yet controlled, binding properties became available. And NCMs cast onto inert supports began to be used widely, this innovation making the manufacturing of ICTs much easier.
Recently, membrane manufacturers have developed new products as substitutes for the nitrocellulose material. While these NCM alternatives have not yet found full acceptance in ICTs, they stand as an example of the separation materials industry reversing the challenge. IVD developers now can test these new materials to discover how they might be used to improve the performance of diagnostics.
Other ICT-Material Advances
IVD developers often express a need for improved materials for ICT components. For example, performing sample preparation off the board defeats one important potential ICT advantage—being able to run the test with minimal, if any, equipment. Test developers recognized that having the ability to separate whole blood, remove particulate contaminants, or otherwise condition the samples would increase the utility of the ICT format. Membrane and filter developers responded with a wide variety of materials, just as they did when challenged to develop new NCMs. Blood separators, sample-filtration materials, and other products were developed in myriad compositions with defined characteristics. IVD developers eagerly put those products to onboard use.
The conjugate support matrix is as crucial to an ICT as the NCM. Not only must it be capable of receiving the liquid conjugate, it must also be able to release the dried conjugate after reconstitution. To enable this to be done efficiently and rapidly, membrane products were developed specifically as conjugate matrices. These newer conjugate-release materials offer significantly better performance than the traditional ones. They are another example of how improvements to an old technology (in this case, fibrous depth filters) can provide a more suitable matrix for assay developers.
The Future
The advance of knowledge regarding the biochemistry of disease processes and the discovery of new biomarkers are opening new avenues for IVD developers to explore. Although the nature of these new markers and of the tests that will be developed to detect them is now unknown, an increasing number of them are expected to follow from proteomics research. In this era of discovery, new technologies will be developed to fulfill the requirements of coming IVDs. More demands will be placed upon diagnostic tests, so it is reasonable to suppose that more functionality will be required from their membrane and filter elements, especially from those that play an active role in the assay.
Membrane and filter manufacturers must be prepared to develop materials for the new IVD technologies that will emerge. Membranes and filters will continue to be an essential part of the IVD development process, both in their supporting role in the preparation of IVD components and in their more central role on the test device.
The question remains unanswered: Will IVD requirements drive development of new and improved membrane and filter products for special applications, or will it be that IVD developers are driven to find creative uses for innovative membrane products?
