Originally Published IVD Technology October 2004
Filters, membranes, and bioseparation equipment and supplies
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| Figure 1. A microporous cellulose nitrate membrane by Sartorius AG (Göttingen, Germany) is used as a lateral-flow membrane for diagnostic tests (click to enlarge). |
Filters used by IVD manufacturers encompass both simple materials (e.g., cellulose paper for liquid absorption or clarification) and complex materials (e.g., membrane adsorbers for selective and quantitative retention of target molecules). Filters are important components in IVDs. They either are an inherent part of the device itself, such as in rapid tests, or provide clean and consistent reagents to build and run IVDs. To provide a better understanding of these materials, this article will review how filters are used in IVDs by listing their properties and functions. Developers, manufacturers, and users of IVDs will find this article helpful in choosing the right filters for specific applications.
Using Filters in IVDs
Reagent Production. Filters have become key materials for purifying, concentrating,
and sterilizing reagents used in IVDs. They are also required for bioburden
testing during reagent production.
A good example is the biotechnological process for producing monoclonal antibodies
(MAB), a key component in many IVD instruments and rapid tests. MAB production
starts in the lab, where the appropriate hybridoma cells are created and selected.
At this stage, several filtration devices are used, from simple syringe filters
to sophisticated multiwell plates with microporous membranes or ultrafilters.
During production, cells are grown in bioreactors that are equipped with sterilizing
filters. This is done to ensure the sterility of all incoming liquids and gases.
Once the titer has reached its optimum level, the fermentation broth is centrifuged
and clarified by using a microporous cross-flow system or a cellulose-based
depth filter.
MABs are then separated from the clarified broth through either conventional
bed chromatography or membrane adsorbers. Further purification, concentration,
and final polishing are achieved with ultrafiltration and membrane flow-through
devices, and extended by gel permeation chromatography. Such steps are needed
to remove the remaining DNA, endotoxins, and large protein aggregates. Like
most other reagents, MABs have already encountered many different filter media
before being used in an IVD assay.
Operating Lab Analyzers. A standard test format in the IVD industry is the laboratory
test instrument. Such instruments use reagents containing highly specific biological
or chemical substances that react with the target analytes and generate a measurable
response. The instruments bring such reagents into contact with defined sample
volumes and measure the resulting change in property, such as a change in the
sample’s color or fluorescence. Such tests are commonly performed on highly
automated instruments in hospital labs, which cover the complete spectrum of
IVDs (e.g., clinical chemistry, immunochemistry, hematology, microbiology, infectious
immunology, and genetic testing).
Operating such test instruments increasingly involves separation filters in
order to attain clean and consistent reagents that are free of particles and
aggregates. Using such filters assures the constant quality of not only the
reagents but also the rinsing and dilution water. Filter devices or cartridges
are used directly inside the instruments.
Building Rapid Tests. Another standard test format is the rapid test. Such tests
are performed on single-use strips, cassettes, or flow-through devices. Although
rapid tests are not always faster than huge clinical analyzers, no instrumentation
is needed to obtain the results.
In the IVD industry, membrane filters are most commonly used in rapid-test strips.
Most rapid tests in lateral or flow-through format have a membrane filter at
the heart of their strip assemblies. In fact, rapid tests would be more appropriately
called “IFDs,” or “in-filter diagnostics,” since most of
the main reactions between the different reagents and the sample take place
inside a membrane filter.
Filter Types
The following descriptions of different filter types used in IVDs, along with
the list of suppliers in the following pages, will help IVD manufacturers choose
the appropriate filters for their particular applications. In general, the structure
and surface chemistry of a filter determine its functionality and suitability
for a specific task.
Filter Papers. Filter papers have a variety of different specifications regarding
thickness, density, gloss, wicking speed, etc. Such papers are made of cellulose
fibers that are randomly laid in a wet process and dried. They are extensively
used in rapid tests as prefilters, wicking reservoirs, and support layers.
Nonwoven Filter Materials. Nonwoven filters are fabricated from synthetic fibers
using methods (other than weaving, knitting, braiding, etc.) and processes such
as dry or wet laying, chemical-, hot-, or spun-bonding, and needle punching.
Nearly all types of fibers can be used to manufacture nonwoven materials (e.g.,
polyester, polyolefin, glass fibers) that are used as prefilters and conjugate
pads. Since nonwoven filters represent many different structures, a supplier
might have up to 70 product families that correspond to the various major applications.
One subcategory is bonded fibers that form a nonwoven structure available in
different shapes other than flat sheets. Such bonded-fiber materials are used
as filters, liquid wicks, and other capillary transfer components to absorb,
transfer, or release liquids in a more controlled manner.
Sintered Beads. Such materials are made by fusing spherical plastic beads. Different
synthetic polymers, mainly polyolefins, are used. Similar to bonded-fiber materials,
sintered-bead materials can be produced in nearly any shape due to the freedom
that the process offers, which sometimes facilitates their automatic handling.
The beads are used as filters, reservoirs, and wicks.
Microporous Membranes. Due to their tight pore-size distribution, membranes
are commonly used as screen filters. This distinguishes them from the materials
described above, which are referred to as depth filters. Membranes are produced
through controlled polymer-precipitation processes that involve evaporation,
solvent exchange, or thermally induced phase separation. Filtration applications
of microporous membranes are well known, the most common of which is sterile
filtration of liquids and gases.
Some small-pore-size polyethersulfone and polyamide membranes are used in rapid
glucose strips. However, their volumes are decreasing as the market is moving
toward electrochemical biosensors that do not use membranes. The most popular
membrane used in IVDs is still the large-pore-size cellulose nitrate membrane
in lateral-flow rapid tests. Its precise structure and highly adsorptive surface
allow for rapid protein binding and easy flow of different reagents, including
large latex conjugate particles (see Figure 1).
Ultrafiltration Membranes. If a membrane’s pores are small enough, the
membrane not only retains particles but also holds back larger molecules. Ultrafiltration
membranes are rated on the basis of nominal molecular weight cutoffs (NMWCO).
For example, a 100-kilodalton (kD) membrane can concentrate and purify antibodies
that have molecular weights greater than 150 kD. At the same time, proteins
or peptides as small as 1 kD can be concentrated and desalted using ultrafilters
with much lower NMWCOs. Such membranes are implemented in devices that operate
under high pressures or in fast tangential flow to reduce fouling and concentration
polarization. Because of this limitation, they are not used in rapid tests (see
Figure 2).
Membrane Adsorbers. Standard microporous membranes undergo some surface-property
changes. The most common changes turn the naturally hydrophobic polymer surface
into a hydrophilic one to improve wettability. This is achieved by adding surfactant
or a hydrophilic coating layer.
Today, further surface modifications may be accomplished by grafting specific
groups onto the main polymeric backbone. The same chemistries that exist in
chromatography gels (e.g., ion exchange) or reactive aldehyde and epoxy groups
are then available on a membrane to selectively bind molecules in large quantities
because of the large surface area. Membranes precoated with ligands such as
metal chelates, protein A, and protein G are also available off the shelf. However,
compared with chromatography beds, high flow rates can be obtained at moderate
pressures due to the large pores and the membrane structure’s rigidity.
Membrane adsorbers are derived from microporous membranes and exhibit a similar
structure.
Choosing Suitable Materials
IVD manufacturers today have a wide choice of filter materials. One recommendation
for selecting a filter material is to have a precise idea of what it should
achieve. Manufacturers should work with their filter suppliers to get a clear
idea of the relation between the material’s characteristics that are given
on the supplier specifications sheet and the expected performance of the test,
such as sensitivity, specificity, signal intensity, etc. This can be the starting
point for a focused selection of filters that are used for a first round of
performance testing.
New Frontiers for Rapid Tests
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| Figure 1. A microporous cellulose nitrate membrane by Sartorius AG (Göttingen, Germany) is used as a lateral-flow membrane for diagnostic tests (click to enlarge). |
While rapid tests represent a small share of the more than $22 billion worldwide
IVD market, they are the major market for membrane filters. Filter manufacturers
as well as filter users should pay attention to the current challenges that
the rapid-test industry is facing.
Increasing Need for Quantitative Results. In general, rapid tests exhibit impressive
sensitivity along with low cost and ease of use.
For example, a standard pregnancy strip can detect down to 5 mIU of hCG hormone
and costs only a few cents to manufacture. Other tests might exhibit even higher
sensitivity. An experimental test by Sartorius AG (Göttingen, Germany)
exploits a silver enhancement method and can detect 50 pg/ml of botulinum toxin
type D. In other words, a simple strip using 150 µl of sample can detect
10–12 g of the toxin.
However, rapid tests do have a major limitation: they deliver only qualitative
results. Because a “yes” or “no” answer detected by the
human eye is often unsatisfactory, IVD manufacturers have been developing rapid
tests that deliver quantitative results, some of which are being commercialized
with some success. The most common readers translate line intensity into analyte
concentration, and use either colorimetric reflectance or a CCD camera to measure
the signal intensity. Other commercially available readers use the emittance
of a fluorescent label that is fixed to conjugate particles. A magnetic reader
for detecting paramagnetic beads was recently introduced. Different manufacturers
are also developing many other technologies.
Depending on the technology, the particular signal that is emitted from a marker
determines to what depth within the membrane a marker will contribute to the
overall signal. As used in colorimetric or fluorescent techniques, visible light
is scattered by the membrane structure and will only contribute when situated
in the top 20–30 µm of the membrane. However, magnetic signals penetrate
the membrane structure more easily and will be less attenuated, giving an integrated,
more accurate signal of the total amount of marker captured in a test line.
While there is a clear goal for IVD manufacturers to develop rapid tests with
reliable quantification, it is not only up to them to reach that goal. For example,
producing lateral-flow membranes that are at the heart of any rapid test and
act as a sophisticated solid-phase substrate and liquid-delivery system puts
high demands on membrane suppliers with respect to membrane quality and consistency.
To get a quantitative signal on a lateral test, all strip components need to
behave in a quantitative manner (i.e., they need to be produced within tight
specifications to deliver the required reproducibility).
Race for New Markers. Pregnancy tests and glucose strips have long dominated
the rapid-test market in terms of sales volume. New markers, though, are needed
to open up new opportunities. Recent discussions among scientists indicated
that the difficulty in finding new single markers might be due to the genetic
heterogeneity among populations. In other words, testing for one single marker
may no longer be sufficient. Tests need to contain a larger panel of markers
to detect only one specific disease or metabolic function.
To test large panels of analytes in one device with one sample is a challenge
for rapid-test manufacturers and their suppliers. Microarrays might play a role
in defining such new test formats. Today, most microarray slides are still made
of surface-treated glass. However, filters and especially membranes are already
contributing to higher-sensitivity arrays due to their 3-D structure, giving
increased binding capacity at the same footprint.
The saying, “Nice things come in small packages,” is not the only
current paradigm in this market. Other needs propelling the development of the
clinical market are workflows with instruments and materials that enable parallel
tasking as well as quantitative output.
Eric Jallerat
and Volkmar Thom, Sartorius AG (Göttingen, Germany)
Copyright ©2004 IVD Technology
