High-quality, biophysical antibody characterization is vital for every program of antibody development: From Discovery, over development and lead selection until batch release. High-throughput ranking of antibody candidates regarding affinity and developability is typically followed by in-depth characterizations of selected candidates to enable data-driven decisions on the final lead candidate. Frequently, the process includes humanizations or other sequence optimizations and, again, a comparison of several antibody sequences.
After lead selection the demand of biophysical assays is shifted to QC and batch-to-batch comparisons. This ensures comparability of produced material. Finally, buffer screens provide important information for formulation and ensure optimal performance of the antibody of choice.
2bind’s biophysical tool box is perfectly set-up to solve bottlenecks in antibody research. It provides valuable and high quality data to streamline the antibody characterization process. For this, 2bind employes state-of-the art instrumentation and over ten years of biophysical expertise.
Affinity, Avidity, and Quantification
Precise information about antibody-target interactions is key for developing therapeutic and diagnostic antibodies. Our versatile tool box ranges from real-time kinetic binding analysis (BLI, GCI, heliX) to steady-state binding assays (MST, Spectral Shift, ITC). As one of the first CROs world-wide, we can also offer real-time binding kinetics on live cells (RT-IC).
Real-time kinetics: BLI, GCI, heliX
Kinetic constants of the antibody-antigen interaction are key parameters that influence the biologic activity of an antibody. Real-time kinetics allow the study of association and dissociation rates of an interaction to targets and off-targets. With BLI, GCI and heliX, 2bind has three complementary technologies available, which differ in physical measurement principle, sensitivity, and throughput.
High-throughput full kinetics for IgGs: GCI
Identify the most suitable variable domains by high-throughput screens of IgGs in real-time kinetics. The waveRAPID module, featuring the GCI technology, allows the full kinetic characterization of IgGs in high throughput (including association and dissociation rates).
Simultaneous binding screens for bispecifics: heliX
Assess whether your bispecific antibody can bind two targets simultaneously with the dual-color photon detector of heliX biosensors and study the influence of density and ratio of the two targets on the binding strength of your antibody.
Real-time kinetics on living cells: heliXcyto
Measure real-time kinetics on living cells captured on nano-scaled cages on heliXcyto chips to solve the most relevant questions for antibodies targeting proteins on cell surfaces: How fast do they bind to target cells and how long do they stay on target cells?
Buffer screens: BLI
Assess the influence of different buffers on your antibody-antigen interaction in BLI. For comprehensive studies with minimal required number of runs, 2bind offers several computer-assisted design strategies (DoE).
Steady-state for tight binders: MST and Spectral Shift
Determine KDs for tight binders from titration curves in MST and Spectral Shift assays. Real time kinetic methods have a hard time with slow off-rates. In these cases, steady-state methods are the better choice. 2bind is equipped for and offers MST and Spectral Shift assays.
Colloidal, Thermal, and Chemical Stability
Besides affinity and biological function thermal, colloidal and chemical stability of an antibody impact the chance of success during drug development. Easily accessible, high throughput assays with low sample consumption and fast turnaround times provide important information to support solid data-based lead selection, the early identification of potential risks and the development of risk mitigation strategies.
Identify and exclude destabilizing Fab domains in nanoDSF
Impaired thermal stability is an indicator for reduced stability of an antibody’s tertiary structure and a risk factor during antibody development. NanoDSF data integrates inflection points of thermal unfolding curves and the onset of aggregation and allows an identification and deselection of IgGs with destabilizing Fab domains.
Sensitive detection of soluble aggregates in DLS
High throughput screens for soluble aggregates are possible in plate format with sample consumption in the single digit µg scale. DLS is faster and detects aggregation with better sensitivity compared to SEC. It is an important tool for an early ranking of antibody candidates.
Stress tests further differentiate antibody candidates
Repeating nanoDSF and DLS after applied stress conditions often leads to further differentiation of antibody candidates. Stress conditions can include chemically induced unfolding, freeze-thaw cycles, thermal stress as well as mechanical stress.
Detect oligomerization and fragmentation in SEC-MALS
SEC-MALS allows the identification and mass-calculation for oligomers, fragments and aggregates. A HPLC for size exclusion chromatography is used together with a multi angle light scattering detector, providing evidence whether a changed retention time is indeed caused by a change in molecular weight.
Self-interaction assays in DLS
Self-interaction of antibodies is a frequent risk factor that is hard to predict from in silico analyses, but easy to measure in DLS. Antibodies with the tendency to self-interact show a diffusion interaction parameter (kD). Ideally, development candidates have the tendency to repulse each other, indicated by a positive kD.
Hydrophobicity measured in HIC
Hydrophobicity leads to a range of problems, including unspecific binding and unfavorable Pharmacokinetics. Retention time on a HIC column is therefore a useful experimental criterion for candidate ranking and lead selection.