Smarter workflow. Earlier insight. Confident decisions. Combining the power of Biacore™ SPR and FIDA™ technologies.

Murthy Narasimha Bandaru, PhD Scientist, Cytiva
Anna Moberg Senior manager, Cytiva

Abstract

An efficient workflow for detailed understanding of protein characteristics is essential to advance drug discovery and protein research. Structural integrity, aggregation behavior, binding kinetics and affinity play critical roles in determining efficacy and reliability of therapeutic candidates and biological reagents. In this context, we present a streamlined workflow that combines Biacore™ SPR and FIDA™ technologies to deliver high-confidence data for protein characterization, quantitation, and interaction analysis.

We recommend the following sequence: Start with FIDA™ to assess structural quality and confirm reagent integrity. Then proceed with Biacore™ SPR technology for real-time kinetics and affinity measurements. Complete with orthogonal in-solution interaction analysis of the prioritized systems. This sequence minimizes rework, clarifies root causes of variability, and enhances data reliability by aligning quality with functional characterization and orthogonal validation.

Through a series of case studies, we demonstrate how the complementary strengths of Biacore™ SPR and FIDA™ technologies reveal critical insights into protein structure-activity relationships. These examples highlight how factors such as solubility, aggregation, and conformational states influence binding behavior—and how integrating both technologies provides a more complete picture of protein functionality and interactions. This unified workflow empowers researchers to make faster, better-informed decisions with greater confidence in the data guiding their next steps.

1. Introduction

In drug discovery and development, detailed protein characterization is essential for understanding target engagement, mechanism of action, and biological activity. It plays a pivotal role in selection and optimization of therapeutic candidates. At the core of these workflows, protein quality is critical to achieve reliable, reproducible, and actionable results. Poor reagent quality can result in inconsistent binding profiles, misleading kinetic data, and reduced confidence in downstream decisions, especially in high-impact areas such as drug development and quality control.

Concurrently, drug discovery is rapidly evolving. Complex targets such as membrane proteins, targeted protein degraders, intrinsically disordered proteins, and various multivalent formats are the new norm in both academic research and drug discovery.

Addressing quality of reagents, proteins, and their interactants early in the workflow is essential for meaningful data across analytical platforms. Reagent quality can suffer from protein misfolding, oligomerization, or partial denaturation. Failure to address these issues can prevent accurate conclusions with any binding analysis. We present an enhanced protein characterization workflow that integrates FIDA™ (flow induced dispersion analysis) with Biacore™ SPR (surface plasmon resonance) technologies to support qualification of reagents, de-risking assay set-up, and provide confident structure activity relationship.

The combined approach offers broader molecular insights and system understanding than either technology alone, streamlines assay development, reduces time spent on poor quality molecules or hits, and improves repeatability. This ultimately advances protein research, accelerates therapeutic development, and brings the treatments to patients faster.

Recommended workflow for protein characterization

For high-quality protein analysis and reproducible assay performance, we recommend a workflow that leverages the complementary strengths of Biacore™ SPR and FIDA™ technologies (Fig 1):

• Reagent quality control with FIDA™ system
  Assess the structural integrity and solubility of both target and analyte using FIDA™ system. Key parameters include hydrodynamic radius (R_h), polydispersity (PDI), and aggregation. Proceed only if reagents meet quality criteria.
• Kinetics and affinity characterization with Biacore™ SPR system
  Perform label-free, real-time analysis of binding interactions to determine kinetic rate constants (k_a, k_d) and affinity (K_D). This step provides detailed insight into interaction dynamics.
• Orthogonal verification with FIDA™ system
  Use FIDA™ system to cross-verify interaction constants as well as structural characteristics of the formed complex. This orthogonal approach boosts confidence in the data and strengthens insights into structure activity relationship (SAR).

Fig 1. Integrated FIDA™ and Biacore™ SPR workflow: FIDA™ quality check establishes solution-state integrity and reagent solubility before detailed interaction analysis using Biacore™ SPR system. Use FIDA™ system to cross-verify affinity and complex size (structure) to enhance confidence in the data and deepen understanding of protein SAR.

In this workflow, orthogonal interaction studies support validation of interaction constants and give important input to SARs. Because FIDA™ measures the solution-state hydrodynamic radius (R_h), it reports on the structural state of proteins and how interactions alter conformation, size, and behavior in native buffers. SPR complements this by offering high-resolution kinetic and affinity data at the interaction site, along with sensitivity for conformational changes that influence binding. The two perspectives are complementary and provide greater insight into the binding event and its impact on the protein’s behavior. This increases confidence that results reflect true biology rather than artefacts.

2. Technologies at glance

Flow induced dispersion analysis (FIDA™)

A small 40 nL plug of fluorescent indicator (either the protein itself with intrinsic tryptophan fluorescence, or a fluorescently labelled protein) is passed through a capillary by pressure driven laminar flow. As it passes through the capillary, the plug disperses by radial diffusion. The detector records a taylorgram of the fluorescent signal vs time. Fitting the taylorgram yields the diffusivity (D), which converts to hydrodynamic radius (R_h) via the Stokes–Einstein equation. Unlike relative hydrodynamic proxies, R_h is an absolute metric which can be confidently compared across labs, reagents lots, and time. This quantitative anchor underpins decision thresholds (monomer vs oligomer, compaction and expansion upon binding) and supports model expectations (experimental R_h vs PDB/AlphaFold) (1, 2, 3).

FIDA™ LD (lambda dynamics) reads two sections of the fluorescence emission spectrum and detects even miniscule changes in the emission spectrum. In combination with Rh measurement, FIDA™ LD is useful to verify affinity. (4, 5)

Fig 2. FIDA™ passes the sample through a thin capillary, tracks Taylor dispersion, and reports absolute hydrodynamic radius in solution. The dispersion profile is dictated by the size and shape of the molecule. Structural changes induced by ligand binding, oligomerization, conformation, complex assembly, and aggregation can all be monitored with just 40 nL of protein. Further, fluorescence readout allows orthogonal binding readout based on changes in fluorescence (FIDA™ LD).

Biacore™ SPR technology

Biacore™ SPR system provides real-time, label-free monitoring of molecular interactions using surface plasmon resonance (Fig 3a) (6). One interaction partner is attached to a sensor chip and the other flows over the sensor surface. Binding events generate a response proportional to the bound mass on the surface with sensitivity down to picograms. The resulting plot, known as a sensorgram, represents binding response over time and allows detailed analysis of interaction characteristics including kinetics (k_a, k_d), affinity (K_D), and active concentration (Fig 3b). Biacore™ system supports a range of other applications such as yes/no binding studies, specificity testing, potency determination, epitope binning, and comparability assessments (7, 8, 9). It’s a powerful tool for confident decision making in complex protein characterization workflows.

Fig 3. (a) Biacore™ SPR system with a (b) sensorgram readout showing real-time association and dissociation of molecular interactions.

Terminology in this note

• Absolute R_h: Hydrodynamic radius derived using first principles by FIDA™ technology.
• PDI: Polydispersity index; <05 is a practical monodispersity guideline in our workflows.
• Spike count: Discrete, high-amplitude events in taylorgrams indicating insoluble/aggregated particulates.
• RU (SPR): Biacore™ SPR resonance units
• R_max: Analyte maximum binding capacity of the surface (RU)
• k_a, k_d: Association and dissociation rate constants.
• K_D: Equilibrium dissociation constant.
• SCK/MCK: Single-/multi-cycle kinetics.

3. Results and discussion

This section presents a series of case studies designed to illustrate the proposed workflow and added value of combining Biacore™ SPR and FIDA™ technology. The theme throughout these case studies is to evaluate reagent quality and its impact on binding affinity and kinetic measurements. The examples span a range of protein systems to highlight how structural integrity, aggregation, and solubility influence experimental outcomes.

3.1 Bruton’s tyrosine kinase (BTK): Functional characterization studies

BTK was selected as a pharmacologically significant but challenging target, due to the complexity involved in generating reliable kinetic data (10). Kinases are assay-sensitive targets, where loss of structural integrity translates to poor or misleading binding data (11). Here, we evaluated an old batch of biotinylated BTK that was stored for six months according to vendor recommendations (–80 °C).

Capture of BTK on Biacore™ Sensor Chip SA

Biotinylated BTK was readily captured on Sensor Chip SA (streptavidin). The resulting sensorgram showed a very nice and even capture injection with a capture level of 8000 RU (Fig 4a). Thus, there was no justification to doubt the integrity of the protein.

BTK binding studies with known inhibitors

Subsequently Biacore™ single-cycle kinetics (SCK) binding experiments with Fenebrutinib and Vecabrutinib were performed. The resulting sensorgrams showed substantially reduced R_max and non-canonical concentration dependence relative to expectations (Fig 4b, 4c). These anomalies suggest that the BTK reagent may have undergone structural degradation or loss of activity, despite showing good capture on Sensor Chip SA.

(a)

Fig 4. (a) Stable capture response of biotinylated BTK on Sensor Chip SA. Biacore™ SCK sensorgrams of Fenebrutinib (b) and Vecabrutinib (c) binding to BTK, showing very low R_max and non-canonical curve shapes.

FIDA™ reagent quality check reveals severe aggregation

To further investigate, the biotinylated BTK sample was analyzed using Fida™ Neo which revealed a severely aggregated population with no discernible monomeric peak in the taylorgram, consistent with loss of protein functionality (Fig 5). This result indicates that the reagent is not fit-for-purpose for quantitative interaction studies and provides an immediate explanation for observed functional anomalies.

Fig 5. FIDA™ taylorgram of archived BTK showing severe aggregation and no monomer peak.

This example highlights the importance and benefits of thorough reagent quality assessment prior to initiating SPR experiments, especially when working with challenging and assay-sensitive proteins like BTK, where structural integrity directly impacts binding reliability.

3. 2 HSA-LMW binding studies

Human serum albumin (HSA) was selected as a model system to demonstrate how structural quality checks using FIDA™ technology can de-risk detailed interaction analysis with SPR. Applying deliberate thermal stress to HSA, such as heating at 90°C for 30 minutes, reveals how subtle structural compromises can alter binding properties. Additionally, a low-molecular-weight (LMW) compound, digitoxin, illustrates how solubility-driven artifacts can be misinterpreted as binding issues. These examples highlight the importance of verifying reagent integrity with FIDA™ system before kinetics and affinity characterization using Biacore™ SPR system.

FIDA™ reagent quality check identifies native HSA as fit-for-purpose

FIDA™ analysis of native HSA across 20 sample readouts yielded an R_h of 3.76 ± 0.03 nm with PDI below 0.05 and a spike count of 0, consistent with a monodisperse, well-folded protein suitable for downstream assays (Fig 6a). This fast (~5 min), nanoliter-scale quality check establishes baseline integrity before proceeding with SPR analysis.

Fig 6. (a) Native HSA quality check: Representative taylorgram; R_h = 3.76 ± 0.03 nm (n = 20), PDI < 0.05, spike count = 0. (b) Overlay of heat-treated HSA FIDA™ results, heat-treated HSA showing broadened, polydisperse profile; R_h > 15 nm.

Heat-treated HSA shows a clearly compromised structure

FIDA™ analysis of heat-stressed HSA 90°C, 15 min. revealed a markedly broadened taylorgram relative to native HSA, with an R_h above 15 nm which indicates increased polydispersity, consistent with unfolding or oligomerization (Fig 6b).

During attachment of HSA onto Sensor Chip CM5, the heat-treated HSA reached comparable surface levels to native but exhibited increased surface stickiness (shown as slower return to baseline during pre-concentration), indicating altered surface presentation even before any binding measurements were attempted (Fig 7).

Fig 7. Overlay of native (orange curve) vs heat-treated (green curve) HSA attachment onto Sensor Chip CM5. HSA heat-treated showed stickiness to the sensor surface during pre-concentration (circled in black).

Biacore™ SPR binding interactions reveal lower binding affinity for heat-treated HSA

After we established the solution-state characteristics and altered immobilization behavior, binding responses were compared. On Biacore™ SPR system, native HSA yielded clean, concentration-dependent binding to LMW analytes, e.g., warfarin (Fig 8a, 8b). In contrast, heat-treated HSA showed reduced R_max and lack of saturation with increased curvature in the association phase and 10-fold weaker apparent affinities versus native (Fig 8c, 8d). The clear and apparent differences in sensorgram shape are consistent with FIDA™ system, which indicated loss of functional site accessibility caused by misfolding or oligomerization, thereby linking directly to reduced SPR binding performance and lowered binding affinity.

Fig 8. (a, b) Sensorgram and affinity plots for Warfarin binding to native HSA and (c, d) heat-treated HSA reveal a 10-fold reduction in binding strength, indicating structural and functional compromise in the heat-treated form.

Revealing compound solubility as a limiting factor

During preparation of the LMW compounds, Furosemide, Warfarin, and Digitoxin for HSA binding studies, all appeared soluble. Digitoxin was visually confirmed to be soluble. However, analysis using FIDA™ system showed the presence of spike features in the taylorgrams, consistent with aggregates or insoluble particulates (Fig 9c). These findings suggest that compound solubility could be a limiting factor in binding studies.

SPR analysis over native HSA surfaces revealed weak and non-reproducible responses, along with non-specific signals on the reference surface (Fig 9a, 9b). These findings show how solubility or aggregation may compromise interaction analysis.

(c)

Fig 9. (a) Sensorgram shows interaction between digitoxin and native HSA. (b) Biacore™ QC plot of binding to reference shows non-specific binding of digitoxin to the reference surface. (c) FIDA™ taylorgram showing sharp spikes, indicating solubility issues with digitoxin in solution.

3.3 Membrane proteins: Structural integrity and functional characterization studies

Characterizing membrane proteins presents unique challenges due to their hydrophobic nature, structural complexity, and dependence on lipid environments for stability and function. These proteins often require specialized expression systems, solubilization strategies, and sample preparation techniques to retain their native conformation and functional activity.

In this example, we showcase the combined use of FIDA™ system for structural quality check and Biacore™ SPR system for functional characterization. This integrated approach provides a more complete understanding of membrane protein behavior, from native structure to binding kinetics.

FIDA™ system confirms the structure and membrane protein integrity

The membrane protein GltTk was purified in two formats: Using dodecyl maltoside (DDM) detergent and styrene-maleic acid co-polymers (SMALP). FIDA™ analysis was used to assess the hydrodynamic radius (R_h) and structural integrity of GltTk (Fig 10). The theoretical R_h of GltTk in its native state, excluding micelle and lipid contributions, is 4.46 nm. Among the SMALP preparations, one batch failed to maintain native-like structure, as confirmed by Fida™ Neo sizing results, highlighting its utility in early-stage quality control and structural determination.

Sample name	Hydrodynamic radius 1 (nm)
GltTk DDM 17.2.23	6.14
GltTk DDM 31.10.22	6.67
GltTk SMALP 17.2.23	N/A*
GltTk SMALP 17.2.23	10.64

Fig 10. FIDA™ characterization of the membrane protein GltTk, purified in detergent (dodecyl maltoside; DDM) and in styrene-maleic acid co-polymers (SMALP), across two batches.

Biacore™ SPR confirms affinity

Following structural validation, DDM-solubilized GltTk was immobilized on Biacore™ Sensor Chip NTA at 10°C, and a kinetics experiments were performed. SCK binding interactions with aspartate and glutamate were measured, yielding affinities in the range of 300–400 nM, consistent with literature values (Fig 11) (12). This confirms both the functional integrity of the membrane protein and the reliability of the assay setup.

Fig 11. Biacore™ SCK analysis of DDM-solubilized GltTk using aspartate and glutamate as analytes. The suboptimal kinetic fit highlights the inherent difficulty in achieving ideal binding data when working with membrane proteins.

Together, the combination of structural assessment and kinetic profiling provided a comprehensive characterization of GltTk—demonstrating how these technologies can overcome the inherent challenges of working with complex proteins like membrane proteins and provide robust insights into their binding behavior.

3.4 β2-microglobulin (β2m)–anti-β2m binding studies

To illustrate the complementarity between FIDA™ system and SPR affinity methods when reagents are well-behaved, we evaluated the binding of β2-microglobulin (β2M) to a monoclonal anti-β2M antibody. This model demonstrates how a reagent quality check with FIDA™ system, followed by kinetic and affinity characterization using Biacore™ SPR system, and cross-verification of affinity constants with FIDA™ technology, provides a robust and orthogonal approach (11). This workflow boosts confidence in the data and supports reliable interpretation of protein interaction characteristics.

FIDA™ reagent quality check of β2m and anti-β2m antibody

Both reagents passed FIDA™ structural quality check. β2m showed an R_h of 1.93 nm (Fig 12a), closely matching the predicted R_h of 1.97 nm from the FIDA™ PDB Correlator using crystal structure 2D4F, indicating a correctly folded monomer in solution. The anti-β2m antibody exhibited an R_h of 5.24 nm (Fig 12b), consistent with a monomeric IgG.

Fig 12. FIDA™ quality check: (a) β2m R_h (1.93 nm) ≈ predicted from 2D4F (1.97 nm). (b) anti-β2m R_h (5.24 nm) consistent with a monomeric IgG.

Biacore™ SPR kinetics and affinity analysis

Biacore™ single-cycle kinetics of β2m on Sensor Chip CM5 with amine-coupled anti-β2m produced clean, concentration-dependent sensorgrams with excellent fit to a 1:1 binding model (Fig 13a). The resulting kinetic constants were k_a = 1.1 × 10⁶ M⁻¹s⁻¹, k_d = 2.4 × 10⁻³ s⁻¹, and an affinity constant K_D = 2.1 nM consistent with prior kinetic measurements.

FIDA™ orthogonal affinity analysis

The in-solution titration series using 640 nm labelled β2M as indicator (at a fixed concentration of 90 nM) and anti-β2M as analyte (ranging from 0-100 nM, diluted in a 3-fold dilution series) produced a clean, single-site binding curve with an apparent K_D of 1.05 ± 0.09 nM, in good agreement with the SPR-derived affinity (Fig 13b). Equilibrium K_D from FIDA™ technology was used as an orthogonal approach to verify the affinity constant and stoichiometry.

(a)

(b)

Fig 13. (a) Biacore™ SPR sensorgram showing excellent 1:1 binding kinetics between anti-β2m and β2m (b) FIDA™ equilibrium binding curve for β2m (indicator) ↔ anti-β2m (analyte) interaction, yielding compatible affinity constants.

4. Conclusions and recommendations

By integrating Biacore™ SPR and FIDA™ technologies into a unified workflow, researchers gain a more holistic understanding of protein behavior—combining structural, conformational, and interaction data in a single streamlined approach. This complementary strategy not only enhances analytical confidence but also accelerates the path from data to decision. Whether optimizing candidates, troubleshooting variability, or exploring novel targets, this workflow empowers scientists to move forward with clarity, speed, and confidence.

The unified workflow for confident protein characterization

Step 1: Early quality certification with FIDA™ technology

• Rapidly assess protein integrity and ligand solubility in native buffers.
• Identify unsuitable reagents early to avoid downstream issues.
• Only fit-for-purpose samples proceed to kinetic analysis.

Step 2: High-precision binding analysis with Biacore™ SPR technology

• Perform high-throughput kinetic and affinity measurements.
• Gain detailed insights into binding mechanisms and interaction strength.
• Optimize assay conditions with confidence in sample quality.

Step 3: Orthogonal verification with FIDA™ technology

• Confirm interaction constants and complex formation via hydrodynamic radius (R_h).
• Resolve anomalies such as aggregation, heterogeneity, or stoichiometry shifts.
• Strengthen data confidence, especially for complex or novel modalities.

This streamlined, iterative workflow minimizes assay development time, reduces resource use, and enables faster, more confident decisions in protein research and drug discovery.

5. References

1. Application note: Assessment of sample quality with every measurement Fida™ Neo fundamentals. Fida Biosystems.
2. Application note: In-solution characterisation of affinity & structural properties of the FcRn IgG1 Fc interaction during product development. Fida Biosystems.
3. Application note: Linking structure and function use PDB data as the baseline for your Fidabio characterization. Fida Biosystems.
4. Willmer P, Emil, Ray KS, Hundahl AC, Marie R, Jensen H. In-Solution Characterization of Biomolecular Interaction Kinetics under Native Conditions. Analytical Chemistry. Published online August 29, 2025. doi:10.1021/acs.analchem.5c02164
5. Willmer P, Hundahl AC, Marie R, Jensen H. Continuous Titration Based Method for Rapid In‐Solution Analysis of Non‐Covalent Interactions. Chemistry - Methods. Published online February 14, 2025. doi:10.1002/cmtd.202400059
6. Data File: Biacore™ 1 series systems- Label-free interaction analysis. Cytiva, CY29857-10Oct24-DF; 2024.
7. White Paper: Biacore™ systems in discovery and early-stage development of biotherapeutic antibodies. Cytiva, CY12800-12Apr23-WH; 2023.
8. White Paper: Biacore™ surface plasmon resonance systems in late-stage development and quality control of biotherapeutic drugs. Cytiva, CY13627-16Nov23-WH; 2024.
9. White Paper: Biacore™ systems in small molecule drug discovery. Cytiva, CY13794-22Feb22-WP; 2022.
10. Willemsen-Seegers N, Joost C.M. Uitdehaag, Martine B.W. Prinsen, et al. Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance. Journal of Molecular Biology. 2016;429(4):574-586. doi:10.1016/j.jmb.2016.12.019
11. Team D. Kinases and Drug Discovery: Protein Kinases as a Validated Drug Target. Drug Discovery World (DDW). Published December 30, 2016.
12. Reyes N, Oh S, Boudker O. Binding thermodynamics of a glutamate transporter homolog. Nature Structural & Molecular Biology. 2013;20(5):634-640. doi:10.1038/nsmb.2548

6. Acknowledgements

We kindly acknowledge Dr. Philip Addis and Dr. Steven Harborne from Sygnature Discovery for contributing with data showcasing the benefit of combining Biacore™ SPR and FIDA™ technologies for membrane protein characterization.

Resources

Explore Biacore 1 series in 3D

App notes, articles, news and more in our SPR solution area

Education

Access Biacore sensor surface handbook (PDF, no registration required)

What is surface plasmon resonance

Free e-learning courses on SPR Learn the basics on how to get started with Biacore™ SPR assays and perform affinity and kinetics measurements.

More information on FIDA(TM) technology