Accelerating Antibody Discovery Using BLI Biosensor Technology

Accelerating Antibody Discovery through Gator® BLI Biosensor Technology Developing and engineering antibodies for diagnostics or therapeutics necessitates comprehensive characterization of an antigen-antibody interaction. Biolayer Interferometry (BLI) technology has greatly helped speed up the process of antibody discovery. Gator Bio’s BLI platforms and its biosensors offer ease of use, reliablity, and high precision analysis when compared with commonly used labeling techniques, such as enzyme-linked immunosorbent assay (ELISA). Information on binding kinetics, concentration, epitope binning, and affinity is key for the identification of potential targets from tag-fee molecules or molecules with widely used tags. Here, we present data using five selected probes, AntiHuman IgG Fc Gen II (HFCII), Anti-Mouse IgG Fc (MFC), NiNTA, Streptavidin (SA); Flex Streptavidin (Flex SA), and Small Molecule Analysis Probes (SMAP), from Gator’s portfolio of biosensors that can significantly increase the throughput for protein/small molecule quantification and characterization of antigen-antibody interactions, thereby advancing the selection of lead diagnostic and therapeutic antibodies. • Precoated and ready-to-use • Enhanced sensitivity and reduced nonspecific binding • Userful for quanitation, kinetics, and epitope bining with BLI technology • Suitable for a vast array of sample type, including crude (cell lysate and media) and purified proteins • Stable over a broad pH range • Can be regenerated or reactivated • Samples can be reused among different biosensors • Next-gen Gator® BLI platform provides a total solution for antibody discovery. • Gator® instruments and biosensors work together to deliver precise, reliable, and reproducible data compared to other competitive BLI biosensors and systems. • Binding kinetics, epitope binning, and affinity applications using Gator® biosensors are able to assess antibody-antigen interactions very efficiently. • Gator® biosensors enable faster and more cost effective discovery of antibodies for the diagnostic and therapeutic market. Indrani Chakraborty, Harsha Agarwal, Vasundhra Bahl, Jessie Peh, and Vivian Nguyen; Gator Bio, Inc, Palo Alto, CA Table 2. Performance at a glance. Figure 3. Reproducibility after 10 regenerations using anti-Mouse Fc biosensors and a serial dilution of mouse IgG standards from 0.01-10 μg/mL. Figure 4. 12x12 antibody epitope binning using Ni-NTA biosensors for over 12 regenerations without the need for Ni2+ recharging. Red = Competition, Yellow = Ambiguous, and Green = Non-competition. Figure 5B. Global-fit analysis using GatorOne software for antibody-antigen interaction, as shown above. KD = 2.27 nM (r2 = 0.99) Table 3. KD measuremnts using the same set of Flex SA biosensors with two different kinetics pairs. The pairs were used alternatingly for over 10 reactivations. Figure 1. Diagram depicting a kinetics assay sensogram obtained from BLI measurements of a ligand binding to its analyte using Gator® pre-coated biosensors. From the GatorOne analysis software, the association constant (kon), the dissociation constant (koff), and the KD and affinity values are obtained. SCHEMATIC OF A KINETICS ASSAY BIOSENSOR FEATURES IV. STREPTAVIDIN (SA) AND FLEX SA BIOSENSORS Figure 2. Quantitation of human IgG with concentrations ranging from 0.3-6000 μg/mL. Table 1. Kinetic parameters for anti-RBD IgG1 and RBD protein for over 20 regeneration cycles. koff, kon, and KD values are within 20X folds of each other. 1. High performance human Fc conjugate-based biosensor that detects all four isotypes of human IgG. 2. Enhanced dynamic range for quantification, kinetics sensitivity, and better regeneration capabilities. 3. Significantly improves productivity, throughput, and accuracy of quantitation and real-time kinetics, resulting in faster lead selection from antibody screening. 1. Gator Bio’s Mouse Fc biosensor detects antibody concentrations (0.02-2000 μg/mL), providing an expanded dynamic range for rapid antibody screening without dilution steps. Exceeding what traditional methods can produce, nextgen biosensor shows no compromise in data quality with even greater sensitivity, providing faster results and significant cost savings in the antibody sector. 2. Gator® next-gen biosenor maintains high sensitivity and reproducibility even after 10 regenerations, making it very cost effective. 1. Functionalized with QiagenTM Tris-NTA and charged with Ni2+ ions for high affinity immobilization of His-tagged proteins; no Ni2+ recharging step needed with use of Regen and Neutral Buffers. 2. Stable immobilization of His-tagged proteins allows for throughput kinetics and epitope binning of antibodies, enabling rapid and continuous quantification witout the need for Ni2+ recharging. Figure 5C. Kinetics characterization of biotinylated TNF-α and anti-TNF-α for over 10 reactivations. Figure 5A. Affinity measurement for protein-protein interactions. Following a baseline measurement in Gator® Kinetics Buffer, biotinylated rabbit IgG was loaded onto SA biosensors followed by association and dissociation of an antigen from 0-500 nM. 1. Captures biotinylated proteins (molar coupling ratio <3) or proteins with AviTagTM. 2. Biotinylated proteins bound to secondary proteins is measured to determine the kon, koff, and KD of antibody interactions. 3. Gator® Flex SA Kit is the first in the market to provide reactivable streptavidin biosensors. The ability to reuse in different applications without losss in performance is desirable for a cost effective research program. 1. Captures small molecules (down to 150 Da), peptides, and biomolecules. Provides high sensitivity and enhanced sigal when needed. 2. Traditional BLI biosensors struggled with small molecule kinetics. Gator® SMAP biosensor exhibits significantly improved performance with greater loading and global fit. 3. Gator® sensograms exhibit clear binding signal and dissociation curves used to calculate kon, koff, and KD while traditional BLI displays no clear signal. Figure 6A-B. Sensogram plots in kinetics study comparing (A) SMAP biosensors on Gator Bio BLI platform versus (B) another BLI platform and their biosensors. Figure 6C-D. Loading capacity of (C) SMAP biosensors on Gator Bio BLI platform versus (D) another BLI platform and their biosensors. A B A B Figure 6E-F. Global-fit analyses comparing (E) SMAP biosensors on Gator Bio BLI platform versus (F) another BLI platform and their biosensors. C D E F C I. ANTI-HUMAN IgG Fc GEN II BIOSENSOR INTRODUCTION II. ANTI-MOUSE IgG Fc BIOSENSOR III. Ni-NTA BIOSENSOR V. SMAP BIOSENSOR SUMMARY Gator® Platform Other BLI Platform Gator® Platform