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Assay Tutorials : Jun 15, 2011 (Vol. 31, No. 12)

Improving Immunoassay Performance

New Tools Aim to Reduce Turnaround Times in Biotherapeutic Development and Production
  • Günther Reichenberger

Immunoassays (often referred to as ligand-binding assays) are critical at many stages in the development and production of biotherapeutics. The most well-established, conventional format when performing an immunoassay is ELISA, renowned for achieving high specificity, even at very low target concentrations or with subtle differences in target molecular structure. However, ELISA requires complex and time-consuming methodologies together with specialist skills in order to achieve quality results. Assay development is also slow, typically demanding at least two to three weeks for development and optimization of each new assay.

Efforts have been made to improve immunoassay performance, such as introducing multiplex platforms to handle large sample volumes, reducing turnaround times, and, therefore, increasing productivity. Numerous kits offer to reduce assay development times in addition to improving reproducibility. Nevertheless, these steps forward are not without their own issues. For example, cross reactivity, cross talk, and matrix interference often present challenges with standard multiplex assays.

Additional pressure for faster development of robust assays stems from the outsourcing trend. Assays that can be easily transferred between labs and used throughout the biotherapeutics workflow, from research through to GxP environments, are needed. In addition to cost pressures, bioanalytical labs are increasingly faced with demands for faster turnaround times, and the need to carry out a large range of analytical investigations, often made difficult by limited sample availability.

A multiapplication alternative to ELISA available in an open format suitable for a broad spectrum of assays, requiring minimal development time and consuming less sample per assay, is becoming necessary to meet industry demands. Miniaturization and increased automation will allow laboratories to cut project timelines, while eliminating user error and fulfilling requests for increased testing.

Case Study: MedImmune

MedImmune had been using ELISAs for process-related impurity measurement, specifically looking at the levels of host cell proteins (HCPs). The company recently identified a need to increase sample throughput and efficiency in order to handle more projects and meet the requirement for more analytical sample submissions.

Following a review of available technologies, MedImmune selected the Gyros immunoassay platform (Gyrolab™ workstation with Gyrolab Bioaffy CDs) as a prospective solution for carrying out assays at nanoliter scale in a simplified, automated format, with the potential for vastly reducing turnaround times.

A head-to-head comparison using both ELISA and Gyrolab to quantify levels of HCP across 38 in-process samples was carried out. The main goals of the comparison project were to simplify sample preparations, develop more robust assays with broad dynamic ranges and high precision, and achieve higher throughput and faster time to results. Evaluation was based on four main parameters including: sample results, turnaround time, analyst hours, and reagent consumption.

Immunoassay Platform

The Gyros platform used (Figure 1) performs assays within nanoliter-scale microfluidic structures in a CD format. Samples and reagents flow through the Gyrolab Bioaffy CD microstructures under the influence of centrifugal and capillary action as the CDs are spun in the Gyrolab xP workstation.

Working at the nanoliter scale means that the consumption of sample and reagents is drastically cut. Apart from obvious cost-saving on expensive reagents, availability of sample can become a limiting factor, especially as regulatory demands drive the need to perform an increasing number of different analyses on each sample.

Each structure is prepacked with a 15 nanoliter volume affinity column containing Streptavidin-coated particles. These columns provide flexibility for use with a variety of specific assays (e.g., sandwich assay and indirect antibody assay). Implementation of microfluidic principles ensures a controlled flow of capture reagents, sample, and detection reagents, so that all samples within a single CD are processed in parallel.

Parallel processing eliminates the occurrence of time-dependent artifacts that may be observed in a typical plate-based ELISA and ensures that samples are processed under uniform conditions. As each individual microstructure equates to one data point, cross talk is also eliminated.

Within the workstation, a laser-induced fluorescence detector records fluorescence on each column. Accompanying control and analysis software is 21 CFR part 11 compliant and gives flexibility in run setup and data analysis, ensuring that assays can be developed and transferred through to GMP and GLP environments.

Gyrolab Viewer software analyzes fluorescent signals within each microstructure, providing a qualitative visual of binding for each individual column (data point). The graphical image enables scientists to see individual binding responses, within one and a half minutes of run completion. This unique functionality provides an ideal support when selecting the best antibody pair during assay development. Using Gyrolab Viewer, up to ten different pairs or conditions can be evaluated in a single CD and compared within an hour. These individual column profiles are automatically stored and can be exported to overlay for more effective comparison.

Results

In order to fully examine the advantages of the Gyros immunoassay platform compared with ELISA, MedImmune carried out a direct comparison between the two methodologies. The initial step in the process was to establish the assay conditions for the Gyrolab xP workstation, including creation of reagents and establishing basic conditions, optimizing conditions for maximum sensitivity, establishing positive and negative controls, and assay qualification.

Whereas, endpoint assays such as ELISA provide a number from which to back calculate a standard curve, the Gyrolab Evaluator software module calculates results from a standard curve, while the Gyrolab Viewer module provides a qualitative visual of binding for each individual column or data point, providing support when selecting the best antibody pair.

Using the Gyrolab platform, assay conditions were established in just one week, and the first assay comparison confirmed a much greater dynamic range than ELISA (Figure 2). Accuracy and precision were assessed and %CV was found to be within acceptable limits across the entire concentration range.

A head-to-head comparison with ELISA using 38 in-process samples was run, and a detailed comparison of the specific HCP assay showed exactly where the significant improvements were achieved with the nanoliter-scale platform.

Whereas just one day of testing, and two hours of analyst time was needed for the Gyrolab process, the ELISA method required eight days, and 50 hours of analyst time.

An advantage was also seen in the increased dynamic range of 4 logs, compared to 2 logs for ELISA, a significant parameter when looking to reduce the number of assay repeats. The sample-prep process was also simplified as all samples could be diluted 1:2, rather than necessitating various dilutions based on each process step as in an ELISA assay.

One of the most powerful benefits experienced by MedImmune was the considerable increase in the efficiency of assay development, with the Gyros platform producing a 90% success rate on the first run compared to 50% with ELISA, and a sixfold increase in throughput.

Overall, the Gyrolab workstation and ELISA were shown to produce corresponding read-outs for process-related impurities. When turnaround time, analyst hours, and reagent consumption were compared however, it was clear that transferring to the Gyrolab workstation decreased sample repeat rates and reagent consumption, and increased sample throughput and analyst productivity (Figure 3).