Epitope Binning: BIN THE FRIDGE!TM

An important step in the engineering of biotherapeutics is to characterize and group a library of monoclonal antibodies by the epitope binding regions generated against a specific antigen. This “epitope binning” enables the maintenance of epitope diversity and provides important information to broaden intellectual property protection. Wasatch Microfluidics is pleased to announce a label free, array-based SPR package that enables high throughput epitope binning of 96 antibodies directly from supernatant using less than 200 µL of each clone, overcoming the sample consumption, throughput limitations, and/or labeling restrictions of current methods. Wasatch has developed a powerful integrated hardware and software platform that delivers the full potential of label-free, high throughput antibody binning, enabling you to … BIN THE FRIDGE!TM

What is Epitope Binning?

In epitope binning, antibodies are tested in a pairwise combinatorial manner, and those that compete for the same binding region are grouped together into bins.

Why Perform Epitope Binning?

In the quest for therapeutic monoclonal antibodies (mAbs), tools that allow an optimal candidate to be selected from a large number of leads can make the difference between a successful program and a clinical failure. A mAb’s epitope correlates with its functional activity, but the in silico prediction of B-cell epitopes is not yet possible, so epitope selection remains an empirical process. Organizing mAbs into epitope families or “bins” can be helpful, because mAbs that target similar epitopes often share a similar function. An epitope bin with functional activity can provide several potential leads to choose from. Conversely, if mAbs from multiple epitope bins exhibit functional activity, this implies different mechanisms of action, which can be advantageous when pursuing an oligoclonal therapy to treat some cancers or infectious diseases where simultaneous targeting of more than one biological pathway may be needed.

Commonly, however, antibody affinity remains the primary selection mechanism. By using affinity for selection, the result may be the selection of a panel of antibodies that bind to the most one or a few epitopes. This epitope bias can be problematic if the functional epitopes are not represented by those selected with the highest affinity.

Best Way to do Epitope Binning?

Previously, epitope binning has required significant time, sample and manpower for experimental setup and data analysis. This has limited its application to small numbers of samples later in the development process. Wasatch has developed a turnkey, high-throughput SPR solution which consists of three integrated components: Flow printing of microarrays, SPR imaging of the microarrays, and custom software tools for experiment setup and data analysis.

Binning Software Tool

The antibody binning software tool was designed to process the massive data sets enabled by experiments run in an SPRi format, where 96 ligands can be tested against 96 analytes. The primary functionalities are:

  • Read in up to 9,216 sensorgrams based on classical or premix binning assay formats.
  • Quickly explore and curate the data. Plotting of injections, ligand spots, or other groups of interactions is possible from any point in the program, enabling the viewing of the raw data alongside the heat map generation, sorting and bin allocations.
  • Define measurement points and generate a heat map showing blocking and sandwiching interactions with a high degree of automation and user control.
  • Sort the heat map into like-behaved antibodies and assign them to bins using custom algorithms and software automation.

Prior to the development of this software tool, the data analysis of even small panels of epitope binning could be a time intensive and laborious process. Now a data set of 9,216 interactions can be binned in minutes!

EB_FIG1_Classic_Premix_Bin
EB_FIG2_BIN-Funnel
MX96-3
EB_FIG7_SW_HeatMap_Vert_noBG

FURTHER READING

WHITE PAPER: Array SPR Applications - Epitope Binning

This white paper describes the importance of determining epitope diversity early in the discovery process. Epitope binning via array-based SPR can be a powerful tool in this effort, as it enables the binning of 96 mAbs in a single automated run, with less than 200uL of each mAb consumed. The paper also presents the custom software Wasatch has developed for rapid processing of these large data sets.

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IBIS WHITE PAPER: Binning of antibodies‐Tandem

This white paper is one in a series that addresses various protocols for binning of antibodies either using sandwich, premix or tandem protocols.

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PUBLICATION: High-Throughput Epitope Binning Assays on Label-Free Array-Based Biosensors Can Yield Exquisite Epitope Discrimination That Facilitates the Selection of Monoclonal Antibodies with Functional Activity

PLoS One 9(3): e92541, Mar. 2014

Y. Abdiche, A. Miles, J. Eckman, D. Foletti, T.J. Van Blarcom, Y.A. Yeung, J. Pons, A. Rajpal
Wasatch Microfluidics, LLC, 825 North 300 West, Suite C325, Salt Lake City, UT 84103, USA. ben@brooks.nu.

Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb’s epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied.

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