Over the years, ELISAs have been the ligand-binding assay (LBA) of choice to support the discovery and development of biologic therapeutics. Although many sensitive, accu-rate and robust ELISAs have been developed, the emergence of new technologies, includ-ing electro-chemiluminescence (ECL), has allowed improvements in the assay’s sensitiv-ity and dynamic range. Since the main differ-ence between ECL assays and ELISAs resides in the detection step, not much improvement is seen in terms of throughput and workflow. The Gyrolab™ platform, manufactured by Gyros AB (Uppsala, Sweden), combines microfluidic technology and f luorescence detection in a fully automated system. In an effort to make the drug development process more efficient and cost effective, during the past 5 years, sci-entists across the industry have started to eval-uate the Gyrolab’s potential for lower sample and reagent consumption, higher throughput and improved sensitivity compared with cur-rent standard approaches (e.g., ELISA), while maintaining good accuracy and precision [1–6].
The instrument has two carousels, one holds microtiter plates that contain the samples and reagents and the other one holds compact disc (CD) microfluidic devices where the ana lysis takes place. A robotic arm injects samples and reagents into the CD. In the CD, liquid is driven by centrifugal force and samples are captured within a microcolumn filled with streptavidin (SA)-coated beads for indirect quantification. Figure 1 illustrates the CD’s structure. The CD is a complex network of microchannels with hydrophobic barriers designed to gate and
control the flow of liquids when the CD is sub-jected to different rotational speeds. In addi-tion, specific microstructures have columns that are prepacked with SA-coated beads designed to immobilize detection-immune complexes (Figure 2). The detection reagent used in this platform is a fluorescent dye, which allows for indirect quantitation of the analyte. The CD’s design also allows multiplexing, since different antibody pairs may be used on each segment (Figure 1), but this capability was not part of our evaluation.
Honda et al. described how centrifugal force is used to deliver precise volumes through the columns [1]. The Gyrolab allows delivery of sam-ple volumes ranging between 0.02 and 1.0 µl (defined by a structure flanked by hydropho-bic barriers). Delivery of such small volumes in a precise way is achieved by injecting a 100% excess volume, the overf low volume is dis-carded through a first slow spin, while a second stronger spin allows the liquid to overcome the hydrophobic barriers and delivers the reagent or sample through the streptavidin column. The ability to use small volumes constitutes one of the main advantages of the system over other platforms because it allows preservation of reagents and samples. Even with the excess vol-ume that is injected and the dead volume needed for the microtiter plate (2.5 µl), the total volume required may be up to twofold lower for samples and tenfold lower for reagents than that required by ELISA and ECL. This is particularly impor-tant for murine studies, where collection of large volumes (>500 µl) may not be possible unless a terminal bleed is performed.
Application of the Gyrolab™ platform to ligand-binding assays: a user’s perspective
In many areas of drug discovery and development, scientists are in a constant search for methods and platforms to reduce assay time and cost. The Gyrolab™ microfluidics platform that we describe here promises to deliver faster ligand-binding assays with lower reagent and sample consumption, while maintaining good accuracy and precision. Due to its limited track record, we evaluated its performance on assays currently used to support pharmacokinetic and immunogenicity studies, and detection of host cell protein impurities in samples from biotechnology processes. This article summarizes our preliminary conclusions about the utility of the Gyrolab microfluidics platform from Gyros AB.
Johanna R Mora†1, Linda Obenauer-Kutner2 & Vimal Patel3
1Bristol-Myers Squibb, Route 206 & Province Line Rd, Princeton, NJ 0854, USA 2Bristol-Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA 3One Amgen Center Drive, 30E-3-C, Thousand Oaks, CA 91320, USA †Author for correspondence:Tel.: +1 609 252 3613 Fax: +1 609 252 7768 E-mail: [email protected]
We have evaluated the use of the Gyrolab plat-form in a variety of LBAs including pharmaco-kinetics (PK), immunogenicity (IMG) and detection of host cell protein impurities. For the case studies presented herein, while the gen-eral observations are noted, the specific data are not presented.
The assay format used in our laboratory to evaluate the platform’s performance on PK assays is similar to a sandwich ELISA: nA biotinylated antibody (antibody ‘A’) specific
to epitope ‘X’ of the drug is added through the common channel to be distributed to all microstructures and immobilized on the streptavidin column;
nAfter two washes through all microstructures, the standards/controls/samples are individu-ally added (by the robotic arms) to each microstructure to be captured by antibody ‘A’;
nMicrostructures are washed and an Alexa 647-labeled secondary antibody to epitope ‘Y’ of the drug (antibody ‘B’) is added through the common channel to be distributed to all microstructures for binding to the analyte.
We compared the results from PK assays devel-oped on the Gyrolab to equivalent colorimetric ELISAs and/or ECL assays, and in most cases
we found the assay on the Gyrolab to be more sensitive than ELISA, but not quite as sensitive as the ECL assay [7]. In all assay formats tested, a main contributor to the improved sensitivity was the reduced matrix effects (tenfold lower matrix effect than ELISA and twofold lower than ECL), which permitted lower sample dilu-tions. We found one of the main advantages of assays developed on the Gyrolab to be their wider dynamic range, on average tenfold greater than ELISA and comparable to ECL. The dynamic range can be further extended by combining CDs of different capacities (20, 200 and 1000 nl) in the same run, with the advantage that the same conditions are used across CDs. The 20 nl CD is used to increase the ULOQ, while the 1000 nl CD is used to improve sensitivity. The extended dynamic range requires fewer sample dilutions, which reduces analyst time at the bench, reagent consumption and errors introduced during sam-ple preparation. In addition, assays run on the Gyrolab were fully automated and more than twofold faster: ana lysis time of 40 study sam-ples (average number run per day) is 2 h on the Gyrolab versus 5 h by ELISA. Since the assay time on the Gyrolab is unattended and ELISAs require the analyst to add reagents and perform washes inbetween, assays run on the Gyrolab allow scientists to spend their time on other tasks. Reagent consumption was, on average, reduced by half. All this while maintaining good accu-racy, reproducibility and correlation with ELISA and ECL. For one of the PK assays developed, the percentage deviation of the standards and quality controls (QCs) to the nominal value were lower than 20%, with percentage coefficients of varia-tion (CVs) lower than 10%, similar to ELISA. A caveat in the evaluation of new technologies is that assay sensitivity is highly dependent on the antibody pairs used. Therefore, not all assays are expected to see an improvement in sensitivity when transferred to the Gyrolab workstation.
Others have agreed with our assessment of the equipment’s pros, but have cautiously stated cons, such as the limited track record, limitation to the labeling systems used (biotin and Alexa 647) and higher cost [8,9]. Although at a first glance the cost seems higher, when one factors in the cost of analyst time, the Gyrolab workstation is ultimately more cost effective than sequential ELISAs and ECL assays, especially since it is a fully automated system.
Another use of the Gyrolab platform is for impurities testing in drug batches; for example, of host cell protein (HCP). The most commonly
Figure 1. Cut-away view of the compact disk showing three full segments, labeled K, L and M, each with eight microstructures.Reproduced with permission from Gyros AB.
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used method for detection of HCP impurities is ELISA. Although accurate, ELISA is labor intensive, has limited detection range, and is frequently subject to matrix effects, especially when high amounts of impurities are present. As a pilot, we investigated the potential use of the technology to detect for HCP impurities in fermentation and in-process samples. Using the same ELISA-based immunoreagents, we success-fully developed a method to detect HCP impuri-ties in these sample types. Most noteworthy, the platform provided an easy approach for method development. The HCP Gyrolab-based assay not only provided us a wider dynamic range (almost 30-fold higher compared with ELISA), but gen-erated results within a 2 h timeframe without significant matrix effect. In the end, overall throughput was significantly improved for the intended purpose of the method.
In addition to the sandwich formats, we also evaluated a bridging format to evaluate the plat-form’s performance on an IMG assay [10]. For this format, the reagents were not added sequen-tially, instead, equal amounts of biotinylated and Alexa-647 labeled drug were preincubated with the antidrug antibody (ADA) control sample and the mixture was then added to each micro-structure for immobilization on the SA column and subsequent detection (Figure 3). The assay on the Gyrolab showed better sensitivity than a nonbridging colorimetric ELISA, but not as good as an equivalent bridging ECL assay, and much lower matrix effects than ELISA (25 vs 0.25% serum). The drug tolerance was supe-rior to ELISA and comparable to that of the ECL assay. We found the assay to have good precision among two different lots of CDs and five different sequential runs (difference <15%) and just like the PK assays, lower sample and reagent consumption and higher throughput. However, Gorman and Finco found intra- and inter-assay precision to be more variable on the Gyrolab IMG assay they developed than its equivalent ECL IMG assay [11]. Therefore, it is always important to determine precision of the assay once you have optimized conditions and ensure that it is appropriate for the use of the assay. Overall, Gyrolab was found to be a good alternative to ELISA and ECL platforms. It is worth noting that we observed differences in assay sensitivity with different monoclonal and polyclonal ADAs, but were unable to attri-bute it to affinity. Therefore, the user may want to explore potential utility of this platform to detect for low-affinity ADAs.
In recent years, automated liquid handlers have been increasingly utilized to improve accuracy and precision in LBAs. Many vendors offer instruments with nondisposable pipette tips for automated liquid handling. The non-disposable pipette tips offer advantages over those of disposable ones such as better accu-racy and precision, especially in low-volume pipetting, and signif icant cost-reduction. However, there are some concerns of ‘carryover’ for nondisposable pipette tips. The Gyrolab workstation is equipped with ten nondispos-able pipette tips, which are used to transfer reagents and samples from the microplates to the CDs. Typically, eight of the tips are assigned to transfer samples (standards, quality controls and test samples) and the remaining two to transfer reagent (capture and detection reagents). The carryover problem may occur with the sample transfer tips, even though the tips are washed between each sample transfer. This is often caused by a generic wash step that is not stringent enough to completely rinse out the sample from the tip. Depending on the assay, the default wash step may not be adequate. During method development on the Gyrolab workstation, appropriate experi-ments must be designed to evaluate whether the carryover issue exists. If found, customized
Key terms
Ligand-binding assay: Technique or method used for the quantification of large molecules (>4 kDa) present in complex biological matrices.
Microfluidics: Manipulation of fluids within structures of small dimensions that hold volumes at the nanoliter scale.
High-throughput: Method that allows users to perform a series of steps at high speed by using robotics and specialized data-processing and control software.
Pharmacokinetics: Study of a drug’s fate within the body following its administration.
Immunogenicity: Ability of a particular substance, such as an antigen or epitope, to provoke an immune response.
Host cell protein: Proteins made by the organisms used in the production of a recombinant product, and therefore considered impurities in the final product.
Gyrolab workstation: Automated instrument for the performance of ligand-binding assays that uses compact disks with built-in microstructures where the assay takes place and automated nanoliter pipetting to reduce method development time and improve experimental execution efficiencies.
Common channel for liquid distribution
Hydrophobic barrier
Capture column(15 nl)
Area (200 nl)
Individual inlet
Volume definition(1000/200/20 nl)
Overflow channelfor excess liquid
Figure 2. Magnified view of a Bioaffy compact disk microstructure showing a portion of the common channel, individual inlet, capture column and hydrophobic barriers.Reproduced with permission from Gyros AB.
product review | Mora, Obenauer-Kutner & Patel
Bioanalysis (2010) 2(10)1714 future science group
methods, including increased washes, multiple wash buffers, and testing various assay buffers (developed by Gyrolab that specifically address the carryover issue) should be developed. Each pipette tip should be independently tested with appropriately controlled experiments to ensure that carryover is minimized.
Control softwareSoftware version 5.1 is more user friendly than previous versions, which required extensive attention to detail. With the previous versions, the user was faced with the task of creating three different documents in order to perform a run: sample, reagent and loading lists; the latter one being a document that describes where to add samples and reagents into the CD. Version 5.1 has a ‘Wizard’ function that overcomes this issue, allowing the user to set up a run in a matter of minutes. Based on a list of conditions and reagents that the user enters, the Wizard generates a loading list for the user and a docu-ment that describes where to put samples and reagents in the plate(s) including the minimum volume that should be added. Although version 5.1 only comes with three different methods to select for the run, each one is meant to be used according to the type of CD; other methods such as the one we used for the immunoge-nicity assay may be requested from Gyros at no additional cost. Gyrolab methods are opti-mized procedures that scientists may develop to run assays. These include liquid transfer proto-cols, spin speeds, and processes for transferring
samples to the CDs. Some of the pitfalls are that none of the software versions allow the user to skip segments on a CD, which may be desired if the user wants to determine reproducibility among CDs without using all the segments on each CD, and that the software does not allow the user to create runs while the instrument is running, but Gyros is considering a request made by several clients to buy the software for installation on other computers.
Use of the instrument in a regulated environmentThe installation qualification and operational qualification package sold by Gyros allows the user to meet the requirements for compliance with 21 CFR Part 11. Little work is left for the user to perform depending on the require-ments set by the user’s departmental standard operational procedure for the instrument.
Regarding the laboratory information man-agement system (LIMS) used for data reduction, for many years there was not an adequate inter-face between Watson and Gyrolab. However, Gyros recently launched control software version 5.2, which has a LIMS interface that works with Watson generic raw data files.
Future perspectiveGyros’ website lists companies such as BMS, Amgen, MedImmune and Merck among those with multiple Gyrolab workstation units in their laboratories, but in many laboratories the evalua-tion of the technology is still ongoing or it is being used to support discovery work. With the launch of the new LIMS interface, we expect more users to start supporting GxP work with the platform.
In summary, the Gyrolab platform worked well in our hands for PK and impurity HCP assays and there is no doubt that over the next few years it may become one of several preferred methods in those areas. Within the IMG field, we anticipate the majority of the industry to wait for others to demonstrate its utility before trying to support clinical studies with the platform.
In addition, USA-based contract research organizations have been cautious about placing assays on the platform until a request is made to analyze samples using methods specifically developed on the platform. This trend is likely to continue until industry becomes more expe-rienced and confident with the technology. Similarly, some groups within the pharmaceu-tical and biotechnology industries still have reservations about developing and validating
Drug–Alexa 647
Antidrug antibody
Drug–biotin
SA bead
Figure 3. The assay format used for the immunogenicity assay.Reproduced with permission from Gyros AB.
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assays for nonclinical and clinical programs on platforms supported by single vendors, and have opted to stick with ELISAs since multiple companies support the reagents and instruments needed for those assays.
AcknowledgementsThe authors would like to thank Karolina Österlund for her assistance during assay development. Karolina Österlund is a senior application scientist from Gyros AB.
Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert t estimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Executive summary
n In our hands, assays developed on the Gyrolab platform offered a broader dynamic range than their equivalent ELISA, especially when combining compact disks.
n In all applications tested, we found three very attractive features: automation, which allows scientists to reduce their time at the bench; shorter ana lysis time, which increases sample throughput; and reduction of sample and reagent consumption.
n Control software version 5.2 has a LIMS interface that allows users to more easily support GxP work.
n In our experience, the quality of reagents has a high impact on assay performance. Therefore, not all ELISAs are expected to transfer well to the platform.
BibliographyPapers of special note have been highlighted as:n of interestnn of considerable interest
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n Detailed description of the compact disc structure and how liquid is controlled within the channels. Also provides information about the Gyrolab™ viewer (software that provides an image of the signal distribution in the microcolumns).
2 Eriksson C, Agaton C, Kange R et al. Microfluidic ana lysis of antibody specificity in a compact disk format. J. Proteome Res. 5, 1568–1574 (2005).
3 Inganäs M, Dérand H, Eckerseen A et al. Integrated microfluidic compact disc device with potential use in both centralized and point of care laboratory settings. Clin. Chem. 51(10), 1985–1987 (2005).
nn First description of the Gyrolab platform and its application to sandwich immunoassays.
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n Provides data that compares the Gyrolab platform to Biacore and conventional ELISA.
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n First report of a bridging immunogenity assay developed on the Gyrolab platform.
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