Friday, September 21, 2012

Automated miniaturization of a homogeneous assay for Immunogenicity testing of biological drug products

In just about a week from now BioTek will be presenting several posters and presentations at MipTec in Basel, Switzerland. Several of those will discuss current assay methods used in the field of biologics for research and development. As a preview to the content that will be presented in one of the posters, here we give a peak at some preliminary data acquired while adapting an AlphaLISA anti-drug antibody (ADA) immunogenicity assay from a 96- to a 384-well microplate format suitable for automated methods.

ADA assays can provide information to help better understand a common challenge faced when evaluating biological drug products; a common immune response in patients is the development of anti-drug antibodies towards the therapeutic drug of interest. While the response is understood to be nearly universal, the extents to which ADAs impact safety and efficacy during treatment vary widely and are difficult to determine due to ADAs being nearly indistinguishable from the therapeutic drug of interest. While several technology platforms exist to measure immunogenicity, a homogeneous bridging assay format provides a simple workflow with high sensitivity and higher-throughput capabilities than other such methods such as solution ELISA based assays. The AlphaLISA assay platform relies on bridging labeled drug antibody by a bispecific ADA (see below).

The AlphaLISA assay platform relies on bridging labeled drug antibody by a bispecific ADA

The assay is constructed by labeling the drug first with AlphaLISA Acceptor beads and also by biotinylating it. The biotinylation serves to allow the drug to bind to Streptavidin-conjugated AlphaLISA Donor beads. In the presence of ADA, the two beads come into close proximity allowing for detection using AlphaLISA technology.

A model system was used in this study to show proof of concept using a mouse monoclonal antibody representative of a biological drug candidate and a goat-anti-mouse antibody as a positive control ADA. The use of liquid handling and dispensing instrumentation, as well as a high-performance microplate reader, allowed for rapid screening of human serum samples (see below).
AlphaLISA Automated Assay Workflow

The robustness of the assay was first determined for comparison to a solution ELISA format (see below). A Z’-factor of > 0.5 is considered to depict robust assay performance. The AlphaLISA assay provided superior performance when compared to the solution based ELISA with Z’-factors of 0.76 and 0.57, respectively. The AlphaLISA assay also provided the added benefit of forgoing an overnight 4°C incubation step required for complex formation during the solution ELISA experiments resulting in considerable time savings. Furthermore, performing the assay in a 384-well microplate format resulted in significant cost savings per data point by minimizing reagent usage.
The AlphaLISA graph

For further information check out the poster at MipTec in its entirety or, in the coming weeks, it will be available at:

By: BioTek Instruments, Peter J. Brescia, Jr., MSc, MBA, Applications Scientist

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