Ask the Expert: Using High-Throughput Analytical Systems to Facilitate Binding-Kinetics Assays

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Critical quality attributes (CQAs) must be characterized thoroughly to establish a biopharmaceutical’s potency, efficacy, and safety. Such characterization involves some of the most difficult analytical activities that are performed in a biologic’s product life cycle. There is great need for analytical tools that can facilitate such studies. In March 2022, Dr. Kalhari Silva (head of scientific research at Custom Biologics) and Dr. Bob Dass (senior scientist at Sartorius) joined BPI to describe how Octet biolayer interferometry (BLI) technology can be applied to analyze biomolecular interactions. Dass provided an overview of its capabilities. Silva explained how Custom Biologics, a contract research organization (CRO), uses the technology to design and optimize assays of antibody (Ab) binding kinetics.

Basic Principles
Dass described the Octet BLI technology as a high-throughput platform for label-free, real-time detection and evaluation of biomolecular interactions. The system dips a row of coated fiberoptic biosensors into samples kept in a SLAS-format plate. The coating is designed to immobilize binding partners for analysis. Upon application, white light shines through the sensor. Some of that light reflects off a reference surface; some of it reflects off the biolayer that forms and dissipates during analyte binding and dissociation. The Octet BLI instrument measures changes in the interference pattern of the reflected light waves in real time to determine analyte concentration, specificity, affinity, and binding kinetics.

Dass presented a sample workflow for an antibody binding-kinetics assay based on a single-concentration affinity screening of an analyte. Often, analysts monitor Ab kinetics using biotinylated antigens and streptavidin-treated biosensors. The Octet BLI system automatically dips sensors — from two up to 96 — into an aqueous buffer to obtain baseline signals, then into microplate wells containing the ligand molecule, which is loaded/attached to the biosensor surface. After a wash step in an appropriate assay buffer, the biosensor is dipped into wells containing the analyte, which binds to the ligand to form a complex. The binding interaction is followed in real time to reveal the association kinetics. Then, the sensor tip is dipped into fresh buffer, and dissociation of the complex is monitored.

The high throughput of the Octet BLI system raises significant advantages for developing kinetics assays. It enables rapid screening of multiple conditions determined by different buffers, metal ions, detergents, and binding pairs. Thus, the system can be used to identify optimal parameters for enzyme-linked immunosorbent assays (ELISAs), surface plasmon resonance (SPR) immunoassays, and other conventional analytical formats.

Sartorius offers multiple sensor coatings for a wide breadth of ligand chemistries. Because samples require no tagging before processing, analysts can study unmodified molecules and prevent damage to high-value samples. The instrument can analyze a wide range of biomolecules and for many applications, including Fc-receptor analysis, vaccine development studies, ligand screenings, inhibition and competition studies, comparability testing for biosimilars, and quality-control (QC) assays. Several models of the technology are available to address different throughput needs.

Case Studies
Silva drew attention to the complexity of biopharmaceuticals and the many kinds of experiments needed to optimize analytical methods for establishing their CQAs. She provided three examples of her company’s efforts to improve and accelerate development of binding-kinetics assays for therapies based on monoclonal antibodies (MAbs).

Fc-Receptor Analysis: In 2019, Custom Biologics was asked to optimize a binding-kinetics assay for a product comprising three types of MAbs expressed using the same host cell line. The developer was transitioning its “MAb cocktail” to a new expression system and needed to compare the Fc-region binding kinetics of MAbs produced by the two platforms. Custom Biologics screened a full panel of Fc receptors using Octet BLI. Silva spoke about her team’s work for assays based on Fc gamma receptor III (FcγRIII) and neonatal Fc receptor (FcRn) ligands, both of which have ties to MAb efficacy and quality.

For FcγRIII, the team began with a “receptor-down” approach based on nickel–nitrilotriacetic acid (Ni-NTA) sensors. Those, Silva explained, enabled use of commercially available histidine-tagged FcγRIII, eliminating the need to modify analytes and ligands before analysis. To ensure assay accuracy, the team used Octet BLI technology to screen for conditions that minimized nonspecific binding (NSB) to the sensors. Analysts tested two MAbs (the same antibody as produced by both expression systems), two buffers, and untreated Ni-NTA sensors, with no ligand load. One buffer resulted in considerable NSB, and regardless of buffer, results from most runs showed negative drift over time. The team compared NSB with and without a FcγRIII load. Those tests generated similar results and led to screening of other conditions, including the use of different blocking agents. The team found conditions that minimized NSB for a given MAb, but those parameters resulted in significant NSB of the other MAbs in the customer’s cocktail.

Silva’s team next tried an “Ab-down” approach based on sensors coated with antihuman fragment antigen-binding (Fab) region CH1, which immobilizes loaded MAbs. However, FcγRIII bound nonspecifically to the sensors with or without a MAb load. Those sensors were replaced with others based on high-precision streptavidin (SAX). That required biotinylation of FcγRIII but reduced NSB of all product MAbs. Silva and her colleagues screened multiple conditions, determining that a high FcγRIII load increased BLI signals. Subsequent reductions in MAb concentrations minimized NSB levels.

Silva observed that development of an assay for FcRn binding required pH determination. Changes in pH can diminish or increase FcRn–Ab binding, which in turn influences Ab half-life and efficacy. Silva’s team used an Octet BLI system to study the effects of buffer pH (6.0–7.2) on BLI responses to MAb association and dissociation. The workflow was similar to that for the FcγRIII assay, with a receptor-down orientation based on biotinylated FcRn and SAX sensors. Product MAbs showed little NSB to the SAX sensors.

Silva noted that FcRn–MAb dissociation can be biphasic depending on assay conditions. The final step of the initial assay protocol showed rapid Ab dissociation followed by a secondary high-affinity reaction that could skew statistical analysis of the results. The team determined that truncating the dissociation step improved assay accuracy and ensured reproducibility.

Macrophage Mannose Receptors (MMRs): Silva’s next case study focused on optimization of assays to compare the binding kinetics and specificity of an originator and two biosimilar MAbs to MMRs. The MAbs all contain mannose residues on their Fc domains. Thus, their MMR binding properties needed to be determined carefully.

Silva’s team sought to compare the MAbs’ susceptibility to selective glycoprotein clearance, one pathway for which is binding to MMRs. Using protein L biosensors and an Ab-down approach, the Octet BLI technology analyzed MMR association and dissociation rates across different concentrations. The system ran samples with and without Ab loads in parallel. The resulting assay performed reliably well and with reproducible results. To confirm the method’s specificity for the given products, Silva’s team later designed another set of experiments in which they applied a commercially available enzyme to deglycosylate the Abs, then analyzed the different MMR concentrations. MMR binding decreased significantly when deglycosylated Abs were loaded, confirming the assay’s suitability.

Vascular Endothelial Growth Factors (VEGFs): The final example involved inhibitors of VEGFs, angiogenic signal proteins that cancer cells leverage to vascularize tumors. Silva’s team needed to compare the binding characteristics of one originator and two biosimilar Abs for VEGF-A isoform 165. Measuring relative binding potency was of particular interest. Doing so would shed light on the biosimilars’ efficacy and the assay’s linearity to the originator.

Octet BLI second-generation amine-reactive (AR2G) biosensors offered the most compatible ligand chemistry. Analysts immobilized growth factors onto the sensors, then performed a typical kinetics-assay workflow using Ab concentrations of 80%, 100%, and 120%. Results showed good linearity and enabled calculation of the MAbs’ half maximal effective concentration (EC50) values. Based on those values, Silva’s team confirmed the biosimilars’ comparability to the originator molecule in terms of potency and efficacy.

Best Practices
Silva emphasized the importance of choosing a suitable biosensor, which requires careful consideration of analyte characteristics, available ligand chemistries, and related needs for immobilizing the binding partners for analysis. Early in assay development, analysts must take care to minimize NSB with sensors to ensure that BLI results will identify a specific binding event. Optimizing assay buffers can help to achieve reproducible data. Considering the many tests involved in optimization, analytical scientists must leverage high-throughput capabilities, such as those of the Octet BLI system, to establish an assay that provides results in the shortest period of time.

Questions and Answers
What factors cause NSB? Possible factors include charged particles derived from cell-culture media or buffer components. Optimizing composition and concentrations of buffers and blocking agents helps to minimize NSB, as do changes in sensor coatings and binding orientations.

How are association and dissociation times determined? Association should be long enough to capture curvature in the data traces — but not so long that the curves flatten completely or introduce a secondary binding effect of the analyte. For many molecules,
5–10 minutes suffices. Rapidly binding molecules (e.g., FcγRIII) might need only one or two minutes. Dissociation time should occur long enough to observe a 5% decrease in dissociation.

What do negative BLI values indicate? On an Octet BLI system, a negative binding value (inverted sensorgram) can represent a directional phase shift in the signal read at the detector and/or a change in ligand conformation. Such values can appear, e.g., during analysis of virions, virus-like particles, or protein complexes. Negative values arising from specific biomolecular interactions produce inverted sensorgrams that show dose-dependent binding responses.

What is the Octet BLI technology’s working range? The system can handle analytes from small molecules (>150 Da) to viruses and other large molecules. The affinity range is about 10 pM to 1 mM.

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