Human antibodies (yellow) bind to antigens on the surface of influenza.Credit: Nanoclustering / SPL / Getty Images
Recently, a pharmaceutical company came to the antibody-discovery specialist, Abveris, with a serious problem. The pharmaceutical company had tried and failed to develop a monoclonal antibody for an upcoming clinical trial. They needed specific, high-affinity monoclonal antibodies (mAbs) with blocking and non-blocking profiles, and needed them in just 11 weeks. Anna Susi Brousseau, Antibody Characterization team lead at Abveris, a division of Twist Bioscience, was confident.
“We often hear about partners who have gone to another contract research organization (CRO) and ended up with very few, or only one, lead clone,” Brousseau says. “We get this single control from that partner, and we’ll assess it against candidates from our discovery pipeline. Usually, we have hundreds of additional lead options to pick from that the other companies couldn’t find.”
Part of the reason why Abveris is able to move so swiftly is its expertise with the Octet platform. These systems, made by the life-sciences supplier Sartorius, utilize bio-layer interferometry (BLI) to monitor biomolecular interactions, label-free and in real time. When compared to other technologies, the Octet platform’s fluidic-free design and higher throughput, allow researchers to run samples more efficiently.
Members of the Abveris team, including Brousseau, were users of the first-generation Octet QK platform. Their use of BLI and other tools help to provide unique insight into the behavior and functionality of large panels of antibody candidates, which translates into the rapid identification of dozens to hundreds of lead options.
The many applications of BLI
BLI is an optical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on a biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern, which is recorded in real time, providing precise and accurate data on binding specificities, analyte concentrations and rates of association and dissociation.
BLI can also be used to compare binding sites in binning assays to determine the region of a target protein to which an antibody binds. Epitope binning studies can increase the likelihood of choosing a lead antibody with the desired biological activity. Cross-competition assays also are performed to identify mAbs that bind similar epitopes to a previously characterized mAb, as in the generation of biosimilars or biobetters. These assays may also be useful in selecting reagents for sandwich or ELISA-type assays, such as those used for biomarker testing or pharmacodynamic assays, to identify good antibody pairs that bind to an antigen simultaneously.
A unique characteristic of BLI is that only analyte binding or dissociation can shift the interference pattern and generate a response profile. Unbound molecules, changes in the refractive index or pH, or other matrix effects do not affect results, making this technology well suited to measurements in crude samples. In addition, BLI is easy to use, making assay development and optimization a much faster process for customer-oriented CROs.
BLI applications are suitable for both biopharmaceutical and academic research, and allow researchers to analyze protein-protein and protein-small molecule interactions including analytes such as proteins, drugs, viruses, nanoparticles or DNA.
One important application is the evaluation of antibodies. If an organization has already discovered antibody candidates, researchers can use BLI to understand if select candidates should be used in clinical trials. They can explore how candidates behave in vitro, namely if they will recognize a target, stay bound to it and exhibit blocking capabilities to inhibit further interactions. Those studies help researchers to predict which candidates might most impact the targeted biological event.
By way of one example, Brousseau cites recent research into a Hepatitis C vaccine. Scientists haven’t yet been able to create a vaccine for this condition and others, like HIV, because of these viruses’ ability to avoid immune responses when the body is injected with attenuated versions of the pathogens.
In one particular experiment, researchers wanted to understand if their antibody would bind to components in the Hepatitis C virus1. If so, it could potentially be used in therapeutics to encourage the immune system to recognize and fight the virus.
The researchers placed Hepatitis C antibodies on the Octet’s biosensor and introduced portions of the Hepatitis C virus and measured binding specificity, the rate of binding and, more specifically, the on- and off-rates, which provide affinity information. The work could one day help inform a Hepatitis C vaccine, and the approach could be applied to any number of other drug development questions.
Benefits over similar technologies
Research scientists use a variety of methods to analyze biomolecular interactions. Fluorescence-based assays, for example, require extra time for sample preparation and assay optimization steps. Labels also pose their own risks, as they can physically interfere with the interaction or the activity in question. The enzyme-linked immunosorbent assay (ELISA) is a widely used end-point assay, but it only reports on steady-state binding affinities and cannot capture on and off rates. Data on weak interactions may also be lost with the multiple washing steps in an ELISA. Label-free monitoring in real time provides a faster and more complete picture of dynamic interactions and helps scientists make data-driven decisions.
Unlike traditional end-point methods that rely on expensive labeling schemes, BLI is label-free. That means it’s much faster to set up and avoids the risk of functional interference from the label itself.
“If you have to biotinylate a reagent, you can get up to a 50% loss of protein throughout multiple processing steps. It’s also harsher on the protein so you don’t know how it affects the integrity of the final product,” Brousseau says.
Labeling can impact results if it is done near the site where the biomolecules are interacting, says Nick Schnicker, director of the Protein & Crystallography Facility at the University of Iowa’s Carver College of Medicine. Labeling also takes time and could decrease yield if it decreases protein stability.
Another advantage is scalable throughput with a processing capacity of up to 96 samples in parallel. This makes BLI an attractive option for high-throughput, multiparameter industry applications.
This is best illustrated, Schnicker says, by comparing the processes of Octet and surface plasmon resonance (SPR), another biosensor technology frequently used to evaluate biomolecular interactions.
“When studying interactions between two molecules, it is common to use different concentrations of a molecule to find the best binding conditions,” Schnicker says. “Using SPR, the molecule would be loaded on to a sensor chip, tested, and then the concentration would be adjusted and the process repeated for each new concentration. With the Octet, researchers can run between two to 96 samples with different concentrations simultaneously.”
“You can get a full titration series of interactions all at one time,” he says. “And independent of the length of your kinetic measurements, you can have replicates complete in parallel. All tests differ, but for setting up an assay and optimizing it, we used to be talking about one to three days for final results. Utilizing BLI, you can typically have it all done in less than a day.”
In addition, BLI is easy to use, and its fluidic-free approach eliminates sample loading or cleaning steps between measurements. Such efficiency provides a shorter time to result, but it also implies cost savings in terms of operator time, materials and machine maintenance.
“You typically don’t have to worry about cleaning things out and things breaking down,” Schnicker says. “That is a big advantage at a core facility with a lot of users."
For researchers, whether at a large core facility or elsewhere, BLI’s many applications and advantages make it a vital research tool. As the demand for specific, high-affinity molecules continues to grow, it will probably become ever more indispensable.