Originally Published IVD Technology October 2005
Filters, membranes, and bioseparation equipment and supplies
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| The asymmetrical design of BTS SP300 membrane from Pall Corp. (Ann Arbor, MI) allows blood plasma to flow to the downstream side of the material without plugging. |
As scientific and economic activity in disease diagnostics and therapeutics continues to accelerate, so does the need for enabling technology platforms that can bridge from theory to practice. In fact, when the events leading to the major medical breakthroughs of the last century are examined in detail, it becomes evident that their commercial applications closely parallel the development of equally revolutionary manufacturing, engineering, and materials technologies.
In many instances, the full benefits of some of the greatest scientific breakthroughs were not fully realized until decades after their discovery, when adequate technological tools became available for their commercialization. It took over 25 years for the discovery of the DNA structure to directly affect pharmaceutical products, and over 30 years for blood fractionation to become the cornerstone of a multi-billion-dollar therapeutic market.
Not surprisingly, the expanded base of knowledge gained through genomics and proteomics research over the last 15 to 20 years has only recently begun to show up in the diagnostics commercial pipeline. New-generation IVD products, such as the Amplicor system by Roche Diagnostics (Indianapolis) for diagnosing infectious diseases, and the UroVysion kit by Abbott Laboratories Inc. (Abbott Park, IL) for early detection of bladder cancer, are good examples of this trend. Yet the limited number of successful product introductions may still point to the existence of a technology gap. Advances in areas such as sample processing and purification, as well as molecular profiling and detection, are still needed to drive the commercial evolution of diagnostic technologies.
The notion that advanced diagnostic tools will emerge from the customary use of the same traditional materials and processes used over the last 20 years simply defies logic. The future of IVD technology cannot be built solely around the same materials and methods of the 1970s and 1980s. The enabling suppliers of today and tomorrow must embrace their role as collaborative technology partners and deliver continuous technological innovations and improvements.
This article highlights some of the recent advances in membrane and bioseparation materials development as it pertains to their traditional role and potential contribution to the advancement of IVD technology.
Advanced Materials for Sample Processing and Purification
Traditional membrane products for diagnostics rely on the ability of membranes to bind biomolecules and to use those immobilized biomolecules for recognition assays. This has brought about a change, first from test-tube-based, radioactive immunoassays to highly sensitive flow-through assays, and finally to the conjugated particle-immunochromatography tests that currently dominate the marketplace. These markets continue to be important, as tests for blood glucose levels, pregnancy, infectious diseases, biomarkers, and drugs of abuse can all be configured on membrane-based platforms.
Advanced separation materials differ from traditional size-exclusion filters in many respects. Through precise manipulation of their chemical and physical characteristics, advanced microporous materials can selectively separate target sample components or unwanted impurities with a higher degree of efficiency. Leukocyte reduction technology from Pall Corp. (Ann Arbor, MI), for example, has been used extensively in the selective separation of white blood cells and other components from transfused and donated blood.
Most of the early efforts in membrane research and diagnostics focused on adjusting the membrane base polymer to attain optimum wettability, as well as affinity and avidity for biomolecules. Nitrocellulose membranes were first introduced to diagnostic one-step particle assays for their broad biomolecule-binding characteristics and flow characteristics. Subsequently, the strong affinity of nylon membranes for nucleic acids and other biomolecules has been successfully exploited to immobilize or capture specific sample components.
Today, challenges associated with detecting multiple biomarkers from complex clinical samples, and the growing need for higher sensitivity and specificity, are driving the development of specialized microporous substrates:
Asymmetric Materials (open pore size upstream; tight pore size downstream). These materials are less prone to plugging under high protein concentrations or with high particle loads, while allowing filtration of complex, difficult-to-filter samples in one step. These characteristics make it possible to separate plasma from whole blood in one step. They also help the processing of larger volumes of high-protein-and-lipid fluids like milk.
Ion-Exchange Membranes. Differential separation based on charge, traditionally the province of column chromatography, can now be incorporated into membrane-based systems, without beads or the need to prepare columns before use. These membranes have been used for the selection or exclusion of nucleic acids or proteins.
Engineered Surfaces. Pall Corp. has developed the Leukotrap Affinity Prion Reduction Filter system based on experiments with altering the surface properties of fibrous materials.
Derivatized Membranes. Chemical groups such as carboxyls, aldehydes, or anhydrides can be used for covalent attachment of active molecules to create “smart” surfaces. These can be used to directly select targets from a complex solution. Using these membranes, antibodies or mimetic ligands can be attached without loss of biological activity.
Sample Preparation and High Throughput
A common bottleneck for biological assays is sample preparation. Almost all diagnostic testing systems require some form of sample preparation before the actual clinical test can be performed. Industry professionals consistently point to sample prep as being the most labor-intensive and time-consuming stage of the process.
As diagnostic tests become more specific and sensitive, the sample-preparation process is likely to grow more complex. Membrane filtration technology is ideally suited for this purpose and offers significant advantages over traditional methods for clarifying and purifying clinical samples. When properly selected and engineered, microporous materials can be integrated to create a streamlined testing platform. In the case of Cepheid’s (Sunnyvale, CA) GeneXpert system, microfiltration technology has been skillfully engineered into an instrument that purifies and analyzes nucleic acids in less than 5 minutes.
Integrating multiwell filtration plates into the clinical diagnostic setting can also play a role in reducing the number of hardware-dependent steps. The use of membrane bottom plates to purify and process samples on a continuous (on-line) mode can eliminate the need for time-consuming centrifugation and liquid-handling steps. Aside from reducing equipment cost, membrane filtration can also result in increased throughput and make a system simpler to operate and maintain. Practically all of the new-technology membranes can be incorporated into multiwell-plate formats.
Molecular Profiling and Detection
An area of increased development activity and innovation is selective separations for the detection of clinically important biomarkers. This involves the targeted isolation and/or enrichment of such specific sample components as cellular subpopulations and clinically significant biomolecules. As stated earlier, traditional purification by size exclusion is being displaced or augmented by specialized surfaces. These surfaces may remove interfering molecules or capture nonabundant analytes critical to molecular profiling and disease diagnosis. Existing methods for sample preparation are expensive, complicated, and time-consuming; IVD manufacturers are searching for new technologies that are more robust, have fewer steps, and are easier to automate.
In addition to sample prep, membranes can also be used for diagnostic applications of genomic or proteomic research. A goal of such research is to identify critical gene sequences or proteins associated with disease or drug reactivity. Glass-based microarrays use a shotgun approach: As many as 30,000 gene sequences are screened for each sample. If sequences or proteins are better tied to disease processes, it is likely that fewer markers will have to be interrogated—perhaps between 10 and 100 features will be needed. A simple visual pattern may be sufficient to confirm a diagnosis or to determine a patient’s likely drug reaction. Configuring these lower-density panels on membrane strips is easy and leads to lower cost and low capital-equipment investments from the end-users. As a result, more patients can gain access to genetic or other biomarker-based tests.
Convergence
Flow-Through and Lateral Flow. New asymmetric membranes can be used as front ends for lateral-flow assays. In this application, the asymmetric membrane separates the sample fluid, and the target analytes are concentrated or enriched before they are transferred to the lateral-flow membrane for immunochromatographic detection. An example of such a material is Pall BTS SP300, which separates plasma from whole blood. When this material is placed on top of conjugate pads and nitrocellulose used for lateral-flow particle detection, assays for plasma targets can be performed from fingerstick volumes of blood.
Membranes and Microfluidics. In point-of-care-testing devices, membrane and bioseparation materials have been combined with microfluidic systems to carry out complex separation and purification steps. Microfluidic systems typically require small volumes of liquid that are particle-free and contain high concentrations of target. A larger sample volume is applied to the membrane, which then transfers the enriched fluid to the microfluidic system. Additional separation or purification can be carried out by the use of derivatized membrane surfaces or other smart membranes.
Membranes and Chromatography. Bead-based columns are widely used in sample preparation. Nearly every kind of separation has been accomplished using column chromatography. However, although large-volume columns are useful for some testing, they are expensive and clumsy when large numbers of samples have to be processed. A simple solution is to configure the columns in a multiwell-plate format. Beads can be added to wells in a filter bottom plate and then treated as any other column. Beads can be dried in the wells for the manufacture of kits.
Design Capabilities
In the early development stages of the diagnostics market, wet chemistry proved to be a convenient and reliable design platform. As a better understanding of the molecular pathways governing disease began to emerge and new analytical methodologies were developed, diagnostic test manufacturers seized the opportunity to commercialize new product platforms and expand their market penetration.
The introduction of membranes as solid supports fueled the creation of the next-generation IVD device—the point-of-care test strip. This new platform, whether in lateral-flow or flow-through mode, proved to be fast, simple to use, and sensitive enough to be of clinical value.
Platform choices are now more varied and include traditional microplate enzyme-linked immunosorbent assay (ELISA) tests; membrane flow-through; lateral flow; membrane spots or stripes; composite multiwell-plate formats, including beads in filter plates; combinations of filter technology; and microfluidics and gene chips. An era of personalized medicine will work hand-in-hand with new drug-discovery techniques to bring to market highly specific drugs that are also targeted for specific patient subpopulations.
Choice of Provider. In the absence of expert technical and engineering support, such promising capabilities are more difficult to realize. Therefore, IVD engineering and R&D personnel must exercise good judgment when selecting a supplier of filtration and bioseparation materials.
Materials. The technical capabilities of the material must be considered with the supplier’s engineering, application, and regulatory expertise in order to bring a successful diagnostic product to market on time.
Conclusion
The field of in vitro diagnostics has, since its early days, been in a dynamic state of development. It now holds an enviable position at the forefront of the healthcare market. With each technological leap, IVD products have become increasingly more sensitive and precise. As ELISA and membrane-based immunochromatography methods transformed the field in the 1980s, the progress in genomics and proteomics research is fueling the evolution of molecular diagnostics today.
New materials and technology platforms have the potential to drive much-needed advancement in IVD technology and help transform the traditional role of clinical testing to one that is more desirable and valuable—to identify pathology before disease symptoms are expressed, and to provide patient-specific information that can be used to optimize treatment.
The recognition of these new material capabilities by IVD scientists and engineers, and the outcome of their collaborative efforts with suppliers of filtration and bioseparation technologies, will ultimately determine whether the next major breakthrough in diagnostics comes within the next few years—or the next few decades.
Ricardo L. Alfonso and Andrew M. Dubitsky, Pall Life Sciences (Ann Arbor, MI)
Copyright ©2004 IVD Technology
