Proteins represent the richest, most diverse category of druggable molecules, with monoclonal antibodies (mAbs) by far the most successful category. The keys to mAbs' success lie in their chemical and biological similarity to the body's own natural disease-fighting arsenal. More than 60 mAb therapies have been approved in major markets, with an additional 500 in development. Global sales of mAbs now exceed $125 billion with revenue projections reaching $350 billion by 2027.

mAb therapeutics cannot be designed from the ground up, atom-by-atom, as with small molecule drugs. Antibody drug discovery involves huge mAb libraries generated from phage display or through immunization. The good news is these libraries are easy to generate. The bad news is that these entries must be screened, hence the bottleneck.

For decades, scientists have used radiolabeling or fluorescence-based methods to identify and quantify molecular interactions but these methods are limited: Enzyme-linked immunosorbent (ELISA) assays, for example, quantify affinity but provide no information on biological activity. Throughput for ELISA is also insufficient for library screening.

An alternative, surface plasmon resonance (SPR), has been around for 30 years but has a reputation for low throughput.

SPR is an optical biophysical method that simultaneously quantifies and characterizes protein-protein binding in real time. In a typical experiment, library antibodies immobilized on a surface are exposed to test antigens. Protein-antigen binding causes changes in the refractive index on the substrate—the readout. Signals are proportional to changes in mass at the surface, which measure the intensity of the interaction.

Potential and limitations

Very few molecular interactions fall outside the capabilities of modern SPR systems, but users should be aware of the method's potential and limitations.

Instrument sensitivity limits the size of molecular analytes, according to Paul Belcher, Ph.D., Biacore Product Strategy Manager at Cytiva. "Since Biacore™ first commercialized SPR, sensitivity has improved more than 40-fold, which has essentially eliminated size restrictions on analytes. Today's instrumentation can routinely detect interactions involving analytes with molecular weights as low as 100 Dalton, or even calcium ions binding to calmodulin.”

Biacore SPR has been used in non-traditional settings, for example to screen reaction mixtures, including hybridoma supernatants, crude organic chemistry reaction mixtures, biological fluids (blood, serum, and plasma), and even to monitor blood levels of therapeutic drugs in cancer patients. For these applications, sampling rate (data points collected per second) is critical. "The greater the sampling rate, the broader the range of kinetics we can reliably resolve, down to extremely weak, very fast interactions."

Microfluidic-based liquid exchange, a feature of SPR systems from Cytiva and others, allows accurate, reproducible resolution of affinities ranging from mM to low pM, and kinetic ranges from very fast transient interactions to interactions with very slow off rates. "These are difficult to accurately measure with well-based systems because the test molecule remains in the well, where it may re-bind," Belcher says.

The throughput question

SPR has been described as an innately low-throughput analysis mode but that characterization is misleading, Belcher says. "Its easy to make a higher-throughput SPR system by simply adding more detection spots and channels, but this always involves sacrificing sensitivity, limiting the types of molecules or interactions you can study, or the quality of the results." This changed, he says, with the introduction of the Biacore 8K platform in 2016. "This instrument is capable of throughput sufficient for early-stage screening of thousands of compounds per day, and provides the sensitivity to support detailed characterization studies in hit-to-lead development."

From its humble beginnings as a commercial instrument platform 30 years ago focused primarily on the characterization of large biologic molecules in research, SPR has evolved into a versatile, accessible technology used throughout the drug discovery workflow in the life sciences, as well as fundamental research and materials science.

"Modern software interfaces, pre-defined methods and protocols, combined with pre-functionalized sensor surfaces and plug-and-play kits, make SPR easy to set up and run," Belcher tells Biocompare. "But the greatest benefit of SPR is that its rich data can guide decision-making, albeit at levels requiring a high level of technical expertise. Higher-throughput SPR is driving the workflow bottleneck from the experiment to data analysis, an issue we are very much focused on solving and which will democratize SPR for users of all levels."

Carterra, which specializes in SPR-based protein characterization, created its HT-SPR™ (high-throughput SPR) platform specifically to address SPR's throughput issues. HT-SPR screens and characterizes mAbs in one step, according to the company.

Carterra’s LSA instrument is based on high-throughput flow printing, which uses microchannels to print up to 384 protein arrays onto the SPR sensor surface, plus 48 reference spots. LSA's bidirectional flow design gets the most from scarce samples by extending immobilization and dissociation times.

The system's capabilities include epitope mapping/binning, kinetics, affinity, quantitation, or any combination of the above in up to 384 assays. Researchers have used this platform to identify COVID neutralizing antibodies, to quality-check transgenic animals, and to validate commercial antibody libraries.

Multi-mode analysis

Combining two or more analytical modes into one method is a standard approach toward designing analyses that are more than the sum of their parts. Liquid chromatography-mass spectrometry (LC-MS) is arguably the most successful example. This strategy works with SPR as well to provide hyphenated methods incorporating SPR plus electrochemistry, HPLC, or MS. An interesting twist on this idea is SPR-microscopy (SPRM), an instrument platform developed by Biosensing Instrument. SPRM combines high-resolution optical microscopy with SPR, providing the typical capabilities of label-free SPR with spatial visualization of real-time affinity and kinetics on whole cells.

One of the knocks on SPR is that its molecular targets must be homogeneous and tethered to the surface of the SPR sensor. Membrane proteins, the targets of perhaps half of all drug discovery and development projects, are notoriously difficult to extract and purify. Even when this is possible, biological activity is quite different in solution or tethered to a sensor surface than the same protein in its membrane-bound state.

SPRM is capable of analyzing interactions of membrane-bound proteins in their original physiologic states, with their native activity. Further, microscopy allows identification and mapping of activity on the cell surface. One of SPRM's unique features is the ability to perform SPR using whole cells as the "ligands." In what amount to SPR cell-based assays, immobilized cells are exposed to the test molecules. The interactions between drugs and target receptors induce morphological changes in the cells, which in turn creates the SPR effect.

"Conventional SPR measures refractive index changes due to the molecular interactions at the sensor surface," says Akemi Ueki, Business Development at Biosensing Instrument. "With SPRM, cellular deformations caused by binding, induces the refractive index changes—this works because SPRM is so sensitive."