By optimizing the diagnostic system rather than its components, product developers are more likely to create a robust test device that can be manufactured at lower cost.
Performance attributes and manufacturing considerations can vary dramatically from one diagnostic test to another, but developing such products is always a complex task. R&D managers who work in companies that develop, manufacture, and sell diagnostic test kits are routinely presented with significant product development challenges (see box, below).
Photo courtesy Kinematic Automation
The experience of the R&D manager with the type of test under development can make a huge difference in the success of a project. A manager who has successfully developed and launched one or more products usually finds it easier to answer the many questions that arise during the product and manufacturing design of subsequent tests. Still, the development process can be daunting, and dealing with the many interactions among discrete product components can be overwhelming. This article offers an overview of how the product development effort can be organized to minimize the amount of time, money, and patience required to complete the process.
The components of lateral-flow diagnostics can be separated into three categories: porous materials, reagents, and housing and lamination materials (see box, below). The components within these categories interact to affect many different aspects of final product performance (see Figure 1).
Figure 1. Schematic cross section of the porous media and reagents that comprise a lateral-flow diagnostic test device.
Effects of Component Interactions
The pervasive influence of component interactions can be illustrated by examining many of the performance attributes of a test for detecting human chorionic gonadotropin (hCG). A particularly good example would be assay sensitivity. Following is a list of some of the components and factors that can influence assay sensitivity.
Modifying some of these components, such as the mass of antibody or detector reagent used per test, can have a predictable and significant effect on product cost. Modifying other test components may have no impact on final product cost but can be extremely difficult, time-consuming, and expensive to investigate.
An example of the latter type of component is the ratio of antibody or antigen molecules per detector reagent particle (specific activity). The diameter of detector reagent particles is fairly well standardized. Most assay developers choose either colloidal gold particles in the range of 30 to 40 nm or colored latex particles in the range of 250 to 350 nm. What is likely to vary from assay to assay, even among those using the same type of detector reagent particle, is the optimal specific activity.
Typically, the relationship between specific activity and achievable assay sensitivity is bell shaped (see Figure 2). However, the steepness of the curve and the optimal ratio are difficult to predict. If an assay developer has the ability and budget to investigate this parameter, it is worthwhile to do so.
Figure 2. Relationship between detector reagent–specific activity and achievable assay sensitivity.
Another example of an expensive and time-consuming component to change and reoptimize is the plastic housing. Once this piece of the diagnostic device has been developed and produced, changes should be considered only as a last resort.
Confounding Variables. Some of the components that can be varied to modify assay sensitivity also affect other aspects of performance. For example, slowing the flow rate of the membrane to increase assay sensitivity will likely extend the time required to obtain a visible signal on marginally positive samples (~25 mIU/ml).
Changing some components can affect a number of performance attributes. For example, increasing the bed volume of the conjugate pad by increasing the pad's thickness will probably augment assay sensitivity by increasing the volume of sample analyzed. However, this approach may also increase the amount of compression between the conjugate pad and the membrane (see Figure 3). Unless it is relieved by changes in the dimensions of the plastic housing, the increased compression will likely also increase the time required for assay completion. In worst cases, excess compression can lower assay sensitivity and result in uneven signal development across the test line.
Figure 3. Schematic of the housing components of a lateral-flow diagnostic test device.
Changes in certain components can be counteracted by changes in other components. For example, using a faster-flowing membrane to satisfy end-of-assay requirements will likely decrease assay sensitivity. This loss in sensitivity can be overcome by increasing the mass of detector reagent or the mass of capture antibody. Making such changes can enable product developers to balance performance against economy.
Test device component categories
Housing and lamination materials
Preventing Nonspecific Binding. Another example of component interactions is the use of blocking agents and detector reagent release agents in the sample and conjugate pads. Two strategies are commonly employed to prevent nonspecific binding of analyte and detector reagent to the membrane.
In the first method, after the capture reagents are applied, nonspecific binding sites on the membrane are blocked by immersion or spray application of a solution containing protein or a highly polar polymer, such as polyvinyl alcohol. However, this technique can displace the capture reagents, especially if they are not optimally fixed by complete drying prior to the blocking step.
In the alternative method, the blocking agents are added to the sample pad. There they cannot, at least initially, interfere with the binding of capture reagents to the membrane. When the sample is added to the test, the blocking agents are resolubilized in the sample pad and carried forward with the sample. Ideally, sufficient blocking reagent is added to the sample pad to prevent nonspecific binding of both the analyte present in the sample and the detector reagent.
Although this approach does not have the disadvantage of affecting the amount of capture reagent bound to the membrane, it can still complicate product optimization. Proteins, polar polymers, and surfactants or detergents can all affect the viscosity of a sample and change the rate at which it flows up the membrane. Additionally, such additives can displace capture reagents from the membrane. By doing so, they may reduce assay sensitivity.
Furthermore, such additives may delay the release of detector reagent from the conjugate pad, because a greater volume of sample may need to pass in order to fully dissolve all of the solids. This requirement may be an advantage if the goal is to increase assay sensitivity. If it is not taken into account at the beginning of the development process, however, it may induce an unacceptable shift in the assay's sensitivity and specificity profiles.
Effects of Product Design. Aspects of product design and manufacturing can deliberately or inadvertently have an impact on the sensitivity of the final product. The length of the membrane strip, especially the percentage of the strip that is downstream of the test line (see Figure 4), may determine the total volume of sample that can be assayed.
Figure 4. Top view of the membrane and reagent components of a lateral-flow immunoassay test device.
Also, the location of the test line relative to the overlap of the conjugate pad will, in combination with the membrane's intrinsic flow properties, determine the instantaneous flow rate of the sample front at the point of detector reagent–analyte complex capture. Once the reagents have been selected, the instantaneous flow rate at the point of capture is the single most important factor controlling binding efficiency.
Effects of Manufacturing Design. The type of reagent application equipment selected determines how best to optimize final product performance. The mode of reagent application (e.g., positive displacement, air-jetting), the type of membrane (lateral flow rate, polyester film–backed, or unbacked), and the dispensing protocol (µl/cm, mg/ml, buffer components, etc.) all affect the width of the capture reagent lines. The width of the lines in turn affects assay sensitivity. The signal becomes more diffuse and harder to visualize as the reagent lines become wider. Once again, it is possible to reduce the width of the capture lines by selecting a slower-flowing membrane, but the impact of this change on end-of-assay requirements must be taken into account.
It is also true that protein binding to nitrocellulose and most or all other types of membranes is maximized by drying the membrane thoroughly (e.g., at 50°–60°C for 3 to 5 minutes in a drying chamber with a high airflow rate). If reagents are applied to a membrane roll using a reel-to-reel system, achieving this level of drying may be more difficult. On the other hand, if product is made in sheets using cards that are 30 cm long, for example, achieving this level of drying may be considerably easier.
Although the previous discussion has not touched on component optimization with the goal of maximizing the robustness or reproducibility of the manufacturing process, this is the fundamental principle behind designing for manufacturability. Unfortunately, until product development is nearly complete, it is normally impossible to predict how variability in one of the components will affect the performance consistency of the finished device.
Specifications for a pregnancy test
Following are the development guidelines given to the research and development manager responsible for designing, developing, and scaling up to manufacturing a hypothetical lateral-flow diagnostic test device for detecting human chorionic gonadotropin (hCG) in urine.
Product Development Plan
The factors discussed above illustrate the complexities involved in product development and device optimization. The examples also suggest why optimizing each component individually and sequentially is likely to be unsatisfactory. Unfortunately, it is impossible to look at and optimize all components simultaneously. The following compromise plan can be tailored to suit most product development objectives.
Identify Manufacturing Equipment. Manufacturing equipment, especially the equipment that is used to apply reagents to the membrane, has a significant effect on the quality and parameters that are used to control the deposition of the capture reagents. Product development should be undertaken using equipment that at least simulates the performance of the equipment that will be used for scale-up and beyond (see Figure 5).
Figure 5. Product developers should use equipment that simulates the performance of processing equipment to be used in full-scale manufacturing. Typical modules for small-scale diagnostic test-strip development and manufacturing (from left): reagent dispenser, slitter, and laminator, by Kinematic Automation Inc. (Twain Harte, CA).
Procure All Necessary Reagents. This is a top-priority activity, whether the reagents are purchased from an outside source or developed internally. If the detector reagent will be purchased, it is of utmost importance to seek out a reputable, high-quality manufacturer. Poor or inconsistent detector reagent quality will not only cause serious problems during product development but is also likely to have disastrous consequences once the product has been scaled up and manufactured. Poor detector reagent quality can lead to uncertain product stability and loss of sensitivity. In the worst cases, the tendency of "naked" detector reagent particles to stick to the first protein they encounter after they have been resolubilized (i.e., the antibody immobilized at the test line) can result in false positives.
Figure 6a. Top laminate of a lateral-flow immunoassay test device.
Procure Samples of Porous Media and Lamination Materials. Specifically, developers should obtain membranes spanning a range of different flow rates (slow, medium, fast), as well as conjugate pads of two or more thicknesses (thin [~0.2–0.5 mm] and thick [~0.8–1.0 mm]). It is also a good idea to source a top laminate (clear polyester film coated with a medical-grade adhesive) so that contact among the various porous media can be achieved without the need to place the strip into a plastic housing (see Figures 6a and 6b).
Figure 6b. A laminated test strip, without housing, showing position of the sample and absorbent pads. Photo courtesy BBInternational (Cardiff, UK).
One of the more difficult components to source may be the plastic backing card onto which all of the other components are laminated. Whereas the cards themselves can be relatively easy to obtain, it may be difficult, and expensive, to find one with just the right dimensions and just the right number of prescored release strips (see Figure 7). At the beginning of the development process, when the primary focus should be on testing and selecting suitable reagents, it should not be a problem if the dimensions of the card are slightly different (± 1 or 2 mm) from what are likely to be the final dimensions.
Figure 7. Schematic of a plastic-backed adhesive film with a prescored release liner.
Apply Capture Reagents. Capture reagents should be applied to the membrane in the approximate position that they will occupy in the final device, accounting for the overlap that will exist between the membrane and conjugate pad. They should be applied in a buffer solution and at a dispense rate that is compatible with the membrane and the reagent application equipment. Membranes with different flow rates and membranes with or without polyester film backing may require significant changes in the dispensing protocol to achieve consistent deposition. The effect of changing the mass of the capture reagent should be examined by changing the concentration of capture reagent solution, not by changing the reagent dispense rate. Dry the membrane appropriately, either completely or under conditions that will simulate final manufacturing conditions.
Test Performance of Membranes and Capture Reagents. Use calibrated liquid suspensions of the detector reagent mixed with appropriately small (~200 µl) volumes of negative and weakly positive sample. Attempt to find a combination of membrane, capture reagent mass, and detector reagent mass that produces acceptable results in terms of sensitivity and specificity, such as the following.
If nonspecific binding (high background on the membrane) is a problem at this point in the product development process, try adding protein (1% bovine serum albumin) to the samples and repeat the experiments.
Assess Assay Performance. Apply the appropriate mass of detector reagent to the conjugate pad(s). Investigate the use of stabilizers (e.g., sucrose or trehalose) and the effect of different drying conditions on release and conjugate stability. Use a top laminate to cover the overlap between the conjugate pad and the membrane to ensure that contact is consistent.
Assess assay performance using the positive and negative samples. Assess the impact on assay sensitivity and specificity of adding proteins, surfactants, detergents, buffer salts (to increase sample molarity), and sodium hydroxide (to bring the pH of the sample solution to approximately 9.5) to the samples.
Develop Working Assay Prototype. Based on the results obtained in the previous set of experiments, pretreat the sample pad material so that it contains an appropriate amount of the additives that enhanced or stabilized performance. Use the top laminate to ensure good contact among the sample pad, the conjugate pad, and the membrane. Reassess assay performance.
Begin Determining Component Robustness. Investigate the effects of varying the following components.
Perform Accelerated Stability Studies. Determine performance stability under accelerated-aging conditions. To reduce the chances of overlooking material-interaction or reagent-stability problems that might take weeks or months to become apparent, developers should study the performance of prototype test strips that have been stored in a desiccated state at elevated temperatures (37°–50°C). Such elevated temperatures are believed to result in faster deterioration of product performance, with each month of accelerated aging equal to three months under standard storage conditions. The strips should be examined at weekly or biweekly intervals for three to six months. In this way, six months of accelerated-aging studies can be used to predict stability and shelf life at 18 months.
Expand Efficacy Testing. Include a range of clinical specimens. Ensure that specimens have been collected and stored in an appropriate manner (bacterially contaminated samples can undermine or delay product development). Test samples that produce unexpected results (false negatives and false positives) using an FDA-approved product to determine whether spurious results are test related or sample related.
Design the Test Housing. Place test strips into the housing and determine performance (see Figure 8).
Figure 8. Typical configuration of a lateral-flow immunoassay device, showing the sample well (round opening) and results (oblong) opening. Photo courtesy BBInternational (Cardiff, UK).
Finalize Component and Manufacturing Specifications. Bring in multiple lots of critical raw materials (normally three or more different lots of membrane, capture reagents, and detector reagent) and manufacture three or more lots of final product. Test performance of each lot against the initial set of specifications.
Throughout the process, it is essential to be looking for the range of effects that varying different components will have on final product performance. It is not sufficient to observe whether the changes in performance are acceptable or unacceptable. As much as possible, the effects of component interactions should be quantified.
In one sense, these interactions can be viewed as being unfortunate because they complicate the product development process. On the other hand, if the various interactions are monitored and quantified, they can be used to the developer's advantage. In instances where interactions are more likely to modulate the performance capabilities of one of the basic components, they can be used to increase the robustness of the manufacturing process. For example, if a slower-flowing membrane is used to enhance marginally adequate reagent sensitivity, it may be possible to avoid product trade-offs.
Applying These Principles
If everything proceeds without any major problems, the process described above should take 6 to 12 months. Occasionally, projects do work out this way. Even if the product development effort doesn't proceed smoothly, feasibility can normally be determined much earlier—perhaps as early as three months after inception—with this process than with traditional approaches. In most cases, product development will not be completed until much later (perhaps 12 to 18 months after inception), because of the time required for stability studies.
The main goal behind this approach is to meter effort and investment so that they do not outpace the reduction in overall product development risk. It is best to tackle the biggest items first—the ones that will be the most difficult and expensive to change later. Only after the biggest challenges have been met does it make sense to start looking at the more flexible components.
Alan Weiss is director of research and development for OEM products at Millipore Corp. (Bedford, MA).