December 1, 2015 (Vol. 35, No. 21)

Adding Labels To Molecules Can Cause Their Interactions To Slip “Off Key.” So Try a Label-Free Approach

Surface plasmon resonance (SPR) sensing platforms reflect well not only on preclinical and clinical development, but also for postmarketing surveillance of approved therapeutics.

SPR sensing is used to measure the binding of an analyte to a target protein immobilized on the surface of a sensor chip. 

SPR technology is all about detecting subtle changes in electron oscillations that occur when biomolecular complexes form on electrically conductive surfaces. Typically, these surfaces have a thin overlay of gold, which can exhibit strong light absorption patterns. These patterns shift due to changes in the refraction when biological molecules bind to an immobilized target on the chip surface.

SPR sensing and a related technique, biolayer interferometry (BLI), allow for label-free detection of even weak molecular binding without the need for fluorescent labeling. These techniques enable fine assessments of both binding affinities and interaction kinetics. For example, real-time data collection produces rich information about binding properties. Also, taking a label-free approach can give a relatively undistorted view of drug-biomolecule interactions, antibody-binding processes, bioprocessing events, and other phenomena.

The Biacore S200, a surface plasmon resonance system from GE Healthcare Life Sciences, is designed to provide outstanding sensitivity. The system can increase the productivity of low-molecular-weight drug discovery programs while delivering reliable affinity, kinetics, or fragment-screening data.

SPR Cleared for Drug Validation

“SPR is unique in comparison with other technologies in that it directly measures molecular interactions without interference from other reagents,” says Fredrik Sundberg, Ph.D., global director of strategic customer relations and market development, GE Healthcare. “The data can be obtained in a fully automated fashion, and the results are exceptionally information-rich.”

Dr. Sundberg notes that over the past 10 years, SPR technology has moved from “nice to have” to “must have” status, with screening and characterization in drug discovery being its primary applications. More recently, SPR technology became essential during the later stages of drug development, and became accepted by the FDA and other health authorities for validating final drug formulations and product potency.

GE Healthcare’s Biacore SPR system and assays became the first label-free systems to receive FDA approval for immunogenicity testing. Repeated injections of recombinant proteins often elicit development of anti-drug antibodies. The immunogenicity of protein drugs is one of the main reasons for the loss of efficacy and the rise of adverse events. The structural characteristics of the biological agent appear to play a major role in development of immunogenicity. “SPR is of great support in characterizing the immune response and predicting clinically significant immunogenicity,” continues Dr. Sundberg.

Researchers at the University of British Columbia (UBC) used Biacore SPR to study binding responses and dissociation rates of neutralizing antibodies against the drug Betasteron, a standard multiple sclerosis therapy. Because SPR is a means of observing binding in real time, and of detecting even relatively weak and transient interactions, the UBC team was able to differentiate between transient and sustained neutralizing antibodies.

UBC researchers were able to collect data that highlighted the possibility of predicting immune response as early as 12 months into the treatment. Importantly, this data permitted the stratification of patients into those who had to discontinue the treatment and those who had the option of continuing.
Sensogram comparison, a recent Biacore innovation, allows for assessment of binding similarities between a reference and samples. Dr. Sundberg points out that superimposition of sensograms may reveal not only if a biosimilar products binds a target receptor, but also if it binds in exactly the same way as the reference protein.

SPR is now routinely included in a battery of analytical techniques to compare the binding properties of a proposed biosimilar with the originator. Another emerging SPR application is production quality control of antiviral vaccines.

“A recombinant vaccine can be a complex virus-like particle with multiple antigenic sites,” explains Dr. Sundberg. “A fully assembled particle must have all sites present and exposed.”

Epitope mapping utilizing Biacore SPR was used by Merck’s research and manufacturing divisions to characterize Recombivax HB®. Multiple antibodies against the surface epitopes showed different degrees of sensitivity to the structural changes occurring during the vaccine production process. SPR provided crucial insights into the particle maturation process and facilitated manufacturing optimization to achieve higher product consistency and stability.

“With continuous guidance by the health authorities and our key opinion leaders,”  concludes Dr. Sundberg, “SPR is well positioned to become a standard technology is the GMP area.”

Biomaterials Development

At Reichert Technologies, SPR systems are developed that utilize an open architecture. Accordingly, these systems can provide robust and flexible platforms for biomolecular interaction analysis.

“All of our fluidics are accessible and can be customized by the user to accommodate a variety of applications,” says Phillip Page, Ph.D., a biosensor sales and field application scientist at the company. “Tubing of larger diameter can be used to run crude lysates, bacterial samples, and cellular cultures.”

Reichert’s standard configuration excels at small molecule and antibody kinetics, with fluidics that can provide outstanding uptime. Additionally, with its plug-and-play capabilities, Reichert’s SPR systems have the added flexibility to carry out a wider variety of research including more specialized experiments.

This capability was used in development of switchable biological surfaces that oscillate between bioinert and bioactive states. The conversion may be caused by an external stimuli whether they are induced thermally, photonically, chemically, or electrically. Also, the change in state can trigger capture or release of biomolecules. Switchable substrates can be tremendously useful in diverse biological and medical applications.

A research team from the University of Birmingham, UK, reported development of surfaces coated with electro-switchable peptides that are capable of regulating biomolecular interactions in response to an applied electrical potential. An SPR experiment was conducted on self-assembled monolayers (SAMs) of peptides that changed conformation upon application of an electrical potential, and thus transitioned from an active to an inactive state.

SPR was used to detect the loss of surface-contained molecules as well as to determine binding kinetics of a test protein to the SAMs. For this experiment, the SAMs were assembled on a Reichert sensor chip, which also served as one of the electrodes. Fluorescently labeled proteins were passed over the sensor chip with or without applying electrical potential. In this fashion, electrically induced conformational changes and binding kinetics could be studied simultaneously.

Reichert offers a variety of flow cells that incorporate technologies such as electrochemistry, fluorescence, and MALDI mass spectrometry. These cells enable increased creativity and scientific utility of SPR.

Another fascinating example of SPR use for biomaterials was developed by researchers  at the University of Connecticut. Reichert’s SPR instrumentation was used to evaluate controlled protein release from hydrogels. Proteins can be effectively trapped into permeable hydrogels using embedded aptamers. Competing oligonucleotides were used to release a trapped protein.

“SPR is really the only technology that can support the real-time analysis of association and dissociation rates of aptamer-protein complexes in the presence or absence of functionalized complementary nucleotides,” concludes Dr. Page. “Various parameters, such as hybridization length, extended aptamer sequences, oligonucleotide concentration and the effect of pegylation, all can be analyzed in a precisely controlled environment. SPR provided for systematic analysis of dynamic molecular interactions, highlighting the great potential of this new promising biomaterial.”

Reichert has ongoing collaborations to further leverage its flexible and robust platform for real-time on-line bioreactor process monitoring, as well as combinations with additional modalities. For example, SPR has been combined with electrospray ionization mass spectrometry.

Rapid Binding Analysis

Fragment-based lead discovery (FBLD) is an increasingly popular methodology for identifying leads in drug discovery programs. SPR shows sufficient sensitivity to generate binding results even with low-affinity compounds. Several thousand compounds could be screened per target in just a few weeks. SPR is highly quantitative and rapidly yields affinity information using very little sample. This is especially useful in FBLD, where a candidate fragment is subjected to various efficiency measurements to help identify the most valuable hits.

“We have developed new technologies that expand the utility of SPR technology into previously untapped applications,” contends Eric Reese, Ph.D., vice president of sales and marketing, SensiQ Technologies. A typical protocol of evaluating binding kinetics of the chemical fragments requires a binding study using multiple dilutions of the same fragment. Dose–response experiments are essential for determining the KD of the interaction.

“SensiQ instruments are designed to eliminate dilution steps, saving reagents and time and ultimately optimizing entire workflows,” adds Aaron Martin, SensiQ’s senior principal scientist.

The innovative injection protocol unique to the SensiQ Pioneer instruments is the OneStep™ method, which exploits Taylor dispersion. The company refers to this harnessing of fluid dynamics as dynamic injection SPR (diSPR™).

The fluid flow path includes a coiled capillary tube, where a sample mixes with a flowing buffer. Samples diffuse into the buffer, creating a continuous 4-log concentration gradient of the analyte. The binding response is collected simultaneously with changing concentrations. The increased data collection rate and continuity of data due to the gradient enhance the extraction of reliable affinity information.

In collaboration with AstraZeneca’s Discovery Sciences group, SensiQ used this method to drive a discovery screening campaign against a poly ADP-ribose polymerase (PARP), a key target in the pharma giant’s drug development and marketing pipeline. The gradient injection identified a secondary weak nonspecific binding site and additional multiple bioactive compounds that had been missed by other SPR instrumentation.

“Obtaining immediate affinity data along the concentration continuum opened new quality control opportunities,” asserts Martin.

SensiQ Technologies has also developed the ability to detect protein aggregation events, an industry first for SPR instrumentation. Biotherapeutic compounds have a propensity to aggregate in a solution. Such aggregates may elicit a strong immune response when administered to patients. Controlling the manufacturing process to decrease aggregation is an intense area of focus for biotherapeutic manufacturers, and SPR can uniquely meet this need.

Martin explains that the real challenge in analyzing protein aggregates lies in the unknown nature and sizes of the formed aggregates. By monitoring diffusion coefficients, scientists from Genentech and SensiQ were able to precisely calculate the aggregation percentage of a production-grade monoclonal antibody. Further work utilizing SPR for manufacturing process optimization is underway with the Genentech collaborators.

Pioneer FE is SensiQ’s flagship surface plasmon resonance system. It is designed to meet the challenges of fragment-based lead discovery by providing rapid affinity data (KD) from primary screens alone, and by enabling prompt identification of fragment candidate actives.

Enhanced SPR Makes Finer Distinctions

BiOptix instrumentation marries the stability and low noise of interferometry with high sensitivity of SPR, resulting in a highly sensitive label-free biosensor (Enhanced SPR, or E-SPR). Binding of analyte to immobilized or captured ligand is monitored in real time by following the phase shift of p-polarized light.

“A layer of gold film, typical for SPR technology, enhances polarization, enabling detection of very low molecular weight compounds,” says Scott Klakamp, Ph.D., vice president, chemistry and biochemistry, BiOptix. “This is especially important in modern FBLD with typical fragment sizes in the 100 to 300 Dalton range.”

FBLD deploys small chemical fragments that may bind only weakly to the desired biological target. Combining them eventually produces the lead of higher affinity.

Dr. Klakamp explains that Enhanced SPR is able to differentiate specific affinities from nonspecific binding even if in the low millimolar range. Analysis of affinity and kinetics of two low molecular weight compounds (157 and 95 Daltons) were tested with immobilized carbonic anhydrase II. The affinities obtained for binding aligned well with previously reported data, with values of 848 nM and 274 nM, highlighting the utility of the technology for screening and qualifying small molecules during drug development.

In addition, BiOptix instruments have unique flow-cell design with two independent flow paths, enabling testing of two unique analytes or two different concentrations of the same analyte injected concurrently. Similarly, simultaneous testing of three solutions reduces the time to find optimal regeneration conditions.

“The affinity and kinetics of a drug binding to its target protein can affect the pharmacokinetics, pharmacodynamics, and efficacy of the drug,” continues Dr. Klakamp. “Our collaborators from Indiana University creatively used E-SPR to detect binding of decoration proteins to a virus-like particle.”

Encapsulation of payload into a synthetic viral particle shows potential in biological applications such as imaging and vaccine development. Exterior presentation of proteins on the surface of viral particles, however, is constricted by difficulties in genetic engineering of coat proteins. Decoration proteins, common in double-stranded bacteriophage viruses, bind at specific locations at the exterior of the capsid and offer an affinity-based alternative to functionalizing viral surfaces.

The team constructed multiple Dec fusion proteins to test the utility of the system in ligand presentation with an eventual goal of translating the findings for cancer immunotherapy. E-SPR was used to analyze the binding affinities of Dec fusions and compare them to a native Dec version. E-SPR was the only method to uncover a second low-affinity binding site on Dec protein, helping resolve contradictions in kinetic binding measurements obtained by other technologies. The team also confirmed that Dec binding is unaffected by genetic modifications, opening the doors for further functionalization.

The BIOptix 404pi surface plasmon resonance instrument has a unique flow cell architecture. It has four needles and four flow cells, and it can be run in two different injection modes. These attributes allow for flexibility in experimental design. The 4 × 1 mode may be used where a reference flow cell is not needed, providing higher throughput in applications such as immunogenicity screening. The 2 × 2 mode may be used to generate high-quality, double-referenced data for kinetic analyses. Also, this mode may allow two independent interactions to be run simultaneously.

Expanding Role for Label-Free Assays

At Pall, the ForteBio division’s Octet and Blitz systems deploy the principles of optical interferometry, specifically, the interaction of light waves. White light is sent down the glass fiber. The light waves reflect from the interface between the glass fiber and the proprietary biocompatible layer and from the interface between the surface chemistry and solution. As the binding complexes start building at the surface of the tip, the interference patterns shift.

“BioLayer Interferometry (BLI) is largely unaffected by the complexity of the samples,” says Dominic Andrada, senior product manager, ForteBio. “BLI users could literally test the samples straight out of bioreactors.”

ForteBio instruments work on a simple “dip and read” principle, with the biosensor tips coated with one of the binding partners dipping into the sample solution without engagement of microfluidics. The tips are consumable, making this technology suitable for applications sensitive to contamination.

Just-in-time bioprocessing quality control is one area that benefits from single-use tips, simplicity of use, and higher throughput. ForteBio offers instruments that can process 1 sample at a time to as many as 96 samples at once. Andrada points to a rapid potency assay as another unmet need in bioprocessing.

Manufacturing process changes and variability in the reagents may affect the potency of final product. BLI could be used to convert relatively simple concentration assays into potency assays by using an algorithm called parallel slope analysis. This methodology has been successfully adopted by the manufacturing quality control department of a national cancer research laboratory that produces vaccines.

“Another unique application for label-free technologies is a comprehensive characterization for genetically engineered antibodies with multiple specificities,” notes Andrada. “It is common to run multiple kinetic experiments to confirm true binding interactions. However, the time commitment to perform the battery of assays for multiple versions of each synthetic antibody is significant. BLI can shorten a 24 hour kinetic experiment to 2 hours.”

Scientists at MedImmune used Octet systems for comprehensive characterization of a novel monovalent bispecific IgG molecule. Such molecules require considerable effort to engineer due to complex pairing of heavy and light chains.

BLI was essential to demonstrate that DuetMab antibodies can concurrently bind two antigens, EGFR and HER2, at the same time. Co-expression of EGFR and HER2 in cancers confers a poor prognosis. The dual inhibition of these receptors promises therapeutic benefits. The binding kinetics of DuetMab antibodies to each antigen and to Fc receptors was indistinguishable from the parental IgG.

This study opens an opportunity for more specific targeting and for lowering toxicities generally associated with administering of a cocktail of two antibodies. BLI data support clinical trials and manufacturing processes, and the data has been submitted to the FDA.

XBiotech uses Octet systems for titer analysis in cell-line development, for assessing recovery and titer in process development and manufacturing, and for release and stability testing and biological potency testing in drug product quality control. BLI titer and binding data were used in an FDA submission to support XBiotech clinical trials.

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