Designing Lateral Flow Assays for Successful Scale-Up and Manufacture
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Lateral flow assays (LFAs) are often developed initially with analytical performance as the primary objective, with emphasis placed on sensitivity, specificity and robustness under intended use conditions. However, an assay that performs well at bench scale will not necessarily translate directly into routine manufacture. As production moves from laboratory methods to reel to reel processing, automated lamination, strip cutting, drying and device assembly, new sources of variation can emerge that affect both manufacturing consistency and assay performance.
Design for manufacture in lateral flow development therefore involves more than scaling an existing strip format. It requires deliberate consideration of how assay architecture, material selection, reagent formulation and process tolerances interact under production conditions. Even small variations introduced during manufacture can affect capillary flow, signal development and overall test consistency.
Previous articles in this series have examined antibody selection, detector systems, matrix effects, and the system level design factors that influence sensitivity and reproducibility. Building on those foundations, this article focuses on how lateral flow assays can be designed for scalable, reliable manufacture, recognising that robust assay performance depends not only on the chemistry of the strip, but on whether that strip can be assembled and controlled consistently under routine production conditions.
Designing for Tolerance and Manufacturing Robustness
A common challenge in lateral flow development is that assays are optimised around nominal dimensions and idealised process settings, with manufacturability considered only after the format has been largely fixed. At that stage, apparently small production tolerances may have larger functional consequences than expected, particularly where the assay relies on narrow pad overlaps, precise line positioning or tight assembly windows.
A more robust approach is to design for tolerance from the outset. In practice, this means asking not only whether the assay works when each component is positioned correctly, but whether it continues to work when realistic manufacturing variation is introduced. Pad overlap, line location, strip width, laminate position, cassette fit, closure pressure and reagent age should all be treated as variable inputs rather than fixed assumptions.
This should also sit within formal design control. Under ISO 13485, manufacturing considerations should be built into design inputs, verification strategy and transfer planning from an early stage. Process failure mode and effects analysis (pFMEA) is particularly useful in identifying which materials, process steps and equipment interactions are most likely to affect assay performance. These outputs can then guide robustness studies and guard banding of critical process parameters, helping define where the true operating limits lie for settings such as lamination pressure, drying conditions, closure height or strip placement.
Reel to Reel Web Handling and Line Registration
One of the most important differences between bench development and production is the introduction of continuous web handling. During reel to reel processing, nitrocellulose must be unwound, transported, aligned and laminated under tension. This introduces positional effects that are largely absent in static benchtop processes, including web wander, tension related movement and dimensional variation across the membrane roll.
These factors can increase tolerance in line placement relative to the final device geometry, even when striping precision itself remains good. This is often more apparent where bench scale development established membrane position against a fixed datum before striping, since that relationship becomes harder to preserve once bobbin fed reel to reel handling is introduced.
In practice, the final position of a test line in the assembled device depends not only on where the reagent was striped, but also on membrane placement on the backing card, nitrocellulose width tolerance, web tracking through the laminator, and how the laminate is later cut and placed into the housing.
In-line vision systems can provide useful control by monitoring stripe position, line width and web registration in real time. They can detect positional drift, missing or irregular lines, and localised out of spec regions before the web is converted further downstream. In some processes, affected regions can then be marked directly on the web so they can be identified later by operators or downstream vision systems and rejected or segregated as needed.
Where these effects are understood early, reel to reel processing can be designed around them through tighter web handling control, suitable datum strategies and in-line monitoring, allowing line placement to remain both functionally robust and manufacturable at scale.
Lamination, Pad Overlap and Assembly Effects
As noted in the previous section, reel to reel processing introduces positional variation that is less prominent in bench scale assembly. During lamination and assembly, this becomes particularly important at component interfaces, where small changes in pad overlap or spacing relative to the membrane can influence fluid transfer and assay kinetics.
Pad overlap influences fluid continuity between components, while the spacing between the conjugate pad and the test line contributes to the effective mixing window available for analyte and detector particles before capture. Changes in these dimensions during design transfer or assembly can therefore affect both liquid transfer and complex formation.
If overlap between sample pad, conjugate pad, membrane and absorbent pad is too small, fluid transfer may become inconsistent or fail entirely. If it is too large, local pooling can occur, changing effective flow behaviour. In blood based assays, overlap design can be particularly important where blood separation materials are used. Excessive overlap of the blood separation pad onto the conjugate pad can reduce the effective surface area available for whole blood cell removal, while insufficient overlap may create a discontinuity in fluid transfer.
Some functional materials also introduce automation challenges because of fragility or low tensile strength. A material that performs well analytically may still be difficult to laminate and convert reproducibly at production scale if it tears easily, distorts during handling or cannot tolerate normal web tension and cutting forces. Material selection should therefore consider not only assay function, but also how reliably the material can be processed.
In practice, these risks can usually be reduced by selecting materials and overlap designs that are inherently tolerant of normal assembly variation, so that fluid transfer remains reliable under routine production conditions rather than only under ideal set up.
Cutting, Strip Placement and Closure Effects
Although strip cutting is often treated as a straightforward converting step, understanding its effect on strip quality and flow behaviour allows it to be controlled more effectively during scale-up. Differences between bench top cutters and automated production equipment can be significant, particularly where blade angle, blade orientation or cutting mechanics differ from those used during development.
Poor blade maintenance can lead to adhesive build up, debris generation and progressively higher reject rates. Dull blades may also compress membrane pores at the strip edge, locally altering capillary flow and contributing to strip to strip variability. In more severe cases, blade movement can introduce shear forces that lift or tear pad materials from the laminate. Frayed edges, adhesive smearing and pad disruption may then affect capillary flow or create variability that is not apparent in early prototypes. Where hardened blade coatings are used, glue should be removed using suitable solvents such as acetone rather than by improvised polishing, which can damage blade geometry.
Mechanical differences between development and production cutting equipment can also alter assay behaviour in less obvious ways. In some transfer scenarios, automated strip cutting can introduce a slight bow in the laminate. If this lifts certain pad materials within the design, it may disrupt fluid transfer and alter the response curve after final assembly relative to bench scale observations.
Strip placement and cassette closure introduce further risk. There must be enough pressure to maintain laminate overlap contact, but not so much that the structure of the materials, particularly the nitrocellulose, is damaged. Excessive compression can produce unusual flow patterns, such as preferential flow along the strip edges or up the centre, resulting in partial line formation or localised hot spots. Misaligned housing pinch points may lift overlaps and interrupt capillary flow, while many off the shelf housings lack effective strip grip features, allowing the strip to move during assembly. Lateral strip movement may leave uneven gaps in the read window, while vertical displacement can cause the strip to bow or become crushed within the cassette. Guard band studies around closure height, pressure and component stack tolerance are therefore valuable for defining a manufacturing window that preserves both device integrity and assay function.
Taken together, these considerations show that cutting and final assembly should be treated as functional process steps rather than simple conversion operations, with equipment set up, maintenance and guard banding used to preserve both strip integrity and assay performance.
Drying Profiles, Reagent Stability and Process Speed
Drying conditions often change substantially when moving from R&D to production, making early drying studies valuable for defining a process window that remains robust at manufacturing scale.
In laboratory development, membranes may be striped in batches, placed into trays and dried overnight in an incubator or drying chamber, for example at 37°C. In reel to reel manufacture, the same reagents may instead pass through drying towers at approximately 45–65°C for only a few minutes before subsequent overnight drying at 37°C. Although both approaches may appear broadly similar, differences in time to drying, airflow, forced air versus passive drying and initial evaporation rate can alter line fixing, line spread, reagent distribution and final performance.
Scale-up can also affect upstream reagent handling. Diluted antibodies, detector conjugates and blocking solutions may be prepared in larger volumes and used over longer periods than in small scale development. It is therefore important to understand how long these materials remain suitable for use once prepared, as changes in stability over the manufacturing window can affect line quality, conjugate performance and final assay consistency.
Scale-up Consideration: Blocking Buffer Stability | |
Potential Issue | When a blocking buffer containing relatively high concentrations of milk derived proteins and other blocking components is prepared at larger scale and used over two days, overnight refrigerated storage can introduce a risk of striping head blockage on the second day. This may not be apparent in bench work, where the buffer is typically prepared and used within a single day. |
Possible Cause | In protein rich systems, cold storage can promote formation of sub visible particulates or protein micro aggregates, even where the buffer still appears clear. Cooling can alter solubility and intermolecular interactions, and these species may not fully redisperse on warming. In a production fluid path, that may be sufficient to obstruct narrow striping lines. |
Development Consideration | This highlights the importance of assessing both functional and physical stability under realistic manufacturing hold conditions, particularly where protein rich buffers are used across extended production windows. |
Similar scale dependent effects can also emerge in conjugate handling and delivery. Parameters such as batch volume, container geometry, mixing conditions, incubation time and purification method can influence particle stability, aggregation behaviour and effective antibody loading, so conjugation processes should be evaluated across intended manufacturing scales rather than assumed to transfer directly from small batch development.
These constraints may also influence process design. In some cases, increasing reel to reel speed from 30 mm/s to 60 mm/s, combined with sufficient drying capacity such as two drying towers, can reduce the time over which sensitive reagents remain in use while maintaining drying performance. In this way, line speed becomes not only a throughput parameter, but also a tool for managing reagent stability and process robustness.
With appropriate hold time studies, drying characterisation and process design, these scale-up effects can be managed systematically, allowing throughput to increase without compromising assay consistency.
Pilot Batches, Process Control and Inspection at Scale
Pilot Batches and Process Challenge
Pilot manufacturing batches provide an important bridge between development, validation batches and routine production. By this stage, critical process parameters and in-process quality attributes should already have been identified through development studies, risk assessment and robustness work. Running pilot batches against draft manufacturing SOPs helps confirm whether those parameters and attributes remain appropriate under production representative conditions, while also exposing ambiguities, hidden assumptions and operator dependent steps before full scale manufacture begins.
Pilot batches also provide an opportunity to train operators early and gather feedback on where the process is difficult to execute consistently, so that issues can be fed back into the design rather than left as downstream manufacturing problems.
Scale-up Consideration: Conjugate Settling | |
Potential Issue | When conjugates based on relatively large particles, such as paramagnetic or latex particles, are held on the machine during extended reel to reel spraying, within reel signal inconsistency may develop over the course of the run, even where bench scale performance appears stable. |
Potential Cause | These larger particles may be more prone to settling in the reservoir vessel during spraying, leading to a time dependent change in the effective particle concentration delivered to the membrane. This may be less apparent at bench scale, where dispense times are shorter and reagent residence time on the machine is limited. |
Development Consideration | This highlights the importance of assessing conjugate suspension stability over the full intended spray duration. Where needed, risk can be reduced through gentle in-process mixing, careful control of bubble formation and smaller aliquots to reduce residence time in the reservoir vessel before use. |
SPC and Routine Process Control
Once critical process parameters and in-process quality attributes have been defined and challenged through pilot manufacture, statistical process control (SPC) can provide an important mechanism for monitoring process stability during routine production. Parameters such as line position, line width, laminate alignment, strip width, closure force or other critical assembly measurements may be suitable for trend monitoring where sufficient data volume exists. Used appropriately, SPC can help detect process drift early, support investigation before defects become widespread, and provide greater confidence that the manufacturing process remains within its intended operating window.
Inspection at Production Speed
Alongside SPC, inspection strategy also becomes increasingly important as throughput rises. Features that are obvious during low speed manual inspection may be difficult to detect reliably on a fast moving production line, particularly where white materials are assembled against white backgrounds, such as an upper wick pad on nitrocellulose. Slight pad shift, edge lift or incorrect placement may therefore be missed unless suitable monitoring is in place. Dyed reference lines can help make positional shifts easier to identify, while more advanced AI enabled vision systems can improve detection of subtle defects such as scratches, fibres, edge lift or slight pad misplacement that are difficult to define consistently using conventional rule based vision alone.
Conclusion
Designing lateral flow assays for scalable, reliable manufacturing requires more than reproducing a successful bench scale format at higher throughput. It requires understanding how materials, process tolerances, equipment behaviour and assembly conditions affect assay performance during scale-up and manufacture.
Successful transfer also depends on structured development activities that help identify where variability is most likely to emerge and how it can be reduced before full scale production. This makes experienced cross functional input essential, since effective scale-up relies on understanding how assay design, materials, equipment capability and process control interact throughout development and transfer.
In practice, successful scale-up depends on combining robust assay design with the manufacturing and process expertise needed to reproduce that performance reliably at scale. When these factors are addressed early, lateral flow assays can be transferred into routine manufacture with a high degree of consistency, control and confidence.
Learn more about Fleet Bioprocessing’s services by exploring our Lateral Flow Assay Development and Transfer to Manufacture pages. Contact us to discuss your project and explore how we can support your assay development goals.
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