Epitope Binning for Next-Generation Biotherapeutic Discovery

The innate ability of antibodies to bind their targets with exquisite specificity and high affinity has been leveraged in the discovery of therapeutic and diagnostic antibodies, so selecting antibodies with appropriate binding characteristics is an important step in early-stage research programs. While an antibody’s epitope largely dictates its biological function, this property can neither be predicted by in silico methods nor shifted rationally by engineering, so it must be selected empirically by screening a myriad of clones routinely produced by modern antibody libraries. Arguably, the epitope is a more relevant screening parameter than affinity, since the latter can be optimized by standard protein-engineering methods, whereas the former cannot. Indeed, it is highly desirable to discover antibodies with unique epitopes that may offer mechanistically differentiated modes of action and intellectual property opportunities. To meet the demand for evaluating these enormous antibody libraries, analytical tools are evolving to accelerate screening while minimizing sample consumption.

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High-Throughput Kinetics with Array SPR

Throughput, speed, resolution, and sample consumption are typically key limiting factors for detailed kinetic characterization early in monoclonal antibody (mAb) discovery campaigns. Here, we show that Array SPR can facilitate the generation of high quality kinetic data from a large panel of clones rapidly and with minimal sample consumption. In this example of a single day’s run, 384 independent kinetic measurements were made on an array comprised of 43 unique mAbs, each immobilized at 8–16 capacities, using a capture approach which does not require purified antibodies. This method required <1 μg per mAb and only 2 μg of the recombinant monomeric antigen. The array format provided well-described and highly reproducible kinetic measurements for clones spanning a 10,000-fold affinity range for their target antigen. These data clearly demonstrate the efficiency and quality of kinetic analysis that is possible using Array SPR.

Retooling Protein Characterization

Protein characterization approaches go hand in hand with analytical instrumentation platforms. This point was expressed in various ways by the presenters at Peptalk: The Protein Science Week, a Cambridge HealthTech Institute event recently held in San Diego. The event’s speakers and presenters also emphasized how protein characterization could advance specific research areas. “Biotherapeutics, ranging from insulin to antibodies to viruses to siRNA, are interesting molecular tools to address a wide range of diseases,” said Wafa Hassouneh, Ph.D., applications scientist, Wyatt Technology. “In the quest to develop effective and robust biotherapies, novel molecules have to be characterized to determine their properties and behavior.”

Antibody Validation Group Urges Multipronged Approach

Although poorly characterized antibodies are blamed for the loss of hundreds of millions of dollars—the bitter fruit of failed or unreliable experiments—dissatisfaction with antibody validation has yet to be adequately addressed. A comprehensive framework for antibody validation across research applications is still lacking. In hopes of stimulating the research community to achieve a higher standard of antibody reproducibility, the International Working Group on Antibody Validation (IWGAV), an independent group of international scientists, issued a set of guidelines. These guidelines, which appeared in Nature Methods, emphasize that antibody validation is best pursued in an application—and context-specific manner.

Antibody Characterization Balances Rigor and Reason

Authentication is becoming a hot-button topic among researchers. For example, researchers who use cells that are meant to possess distinct characteristics have been making an issue of cell-line authentication. With increasing frequency, researchers who use antibodies are raising authentication concerns of their own. These researchers are insistent that antibodies be validated for function and purpose.

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