Biacore SPR for Antibody Affinity Measurement

What is Antibody Affinity?

Antibody affinity refers to the strength of the interaction between a single antigen-binding site of an antibody and its corresponding antigen. It is quantitatively expressed by the dissociation constant (Kd), which represents the equilibrium between the antibody-antigen complex and the dissociated components. A low Kd value indicates a high-affinity interaction, signifying a strong binding between the antibody and antigen, whereas a high Kd reflects weaker binding.

It is important to distinguish affinity from avidity, a related but distinct concept. Avidity describes the overall binding strength in multivalent interactions, where multiple antibody binding sites contribute to the interaction. This becomes particularly significant when antibodies engage with antigens in a multivalent or complex form, as is often seen in immune responses or when engineered antibodies are utilized for therapeutic purposes.

The Role of Affinity in Drug Discovery

In drug discovery, the affinity of an antibody plays a central role in determining its therapeutic potential. High-affinity antibodies are more likely to bind with greater specificity to their target antigen, making them effective at lower doses and minimizing off-target effects. This is particularly important in monoclonal antibody therapies, where high specificity reduces the risk of adverse immune reactions and ensures that the therapeutic antibody acts predominantly at the intended target site.

Furthermore, antibody affinity impacts the pharmacokinetics of the drug. Antibodies with higher affinity tend to have longer half-lives due to prolonged binding and slower dissociation from their targets. This is particularly beneficial for chronic conditions, where sustained therapeutic levels of the antibody are necessary for efficacy.

Why Choose Biacore SPR Technology?

Biacore technology is based on the principle of Surface Plasmon Resonance (SPR), a powerful, real-time analytical technique that allows researchers to measure the binding interactions between molecules without the need for labels. In an SPR experiment, a molecule (such as an antibody) is flowed over a surface that has been modified to immobilize another molecule (typically an antigen). As the antibody binds to the antigen, changes in the refractive index are detected by the Biacore system, providing a direct measure of binding events.

This label-free approach has become indispensable in the field of biosensor technology, offering numerous advantages for antibody affinity measurement:

• Real-time kinetic analysis: Provides immediate insights into binding events as they happen, including both association (binding) and dissociation (unbinding) phases.
• High sensitivity: Detects even low-affinity interactions and works well with low concentrations of analytes.
• Non-invasive: Maintains the natural structure and function of biomolecules, allowing the study of interactions in a native state.
• Quantitative measurement: Directly measures binding events, yielding precise values for affinity constants, dissociation rates, and association rates.

These features make Biacore a powerful tool for studying antibody-antigen interactions, including affinity measurements, kinetic profiling, and more.

How Biacore Measures Antibody Affinity?

The SPR Experiment: Step-by-Step Process

Biacore systems measure antibody affinity by analyzing real-time binding interactions between antibodies and their antigens.

• Immobilization of Antigen: The antigen is immobilized on a sensor chip, typically using methods such as amine-coupling or thiol-coupling, depending on the nature of the antigen and the desired binding conditions.
• Injection of Antibody: The antibody is then flowed over the surface of the sensor chip. As the antibody binds to the antigen, the refractive index at the surface of the sensor chip changes, which is detected by the Biacore system.
• Association and Dissociation Phases: The Biacore system measures both the association phase (when the antibody binds to the antigen) and the dissociation phase (when the antibody unbinds from the antigen). These data are used to calculate the association rate (ka) and dissociation rate (kd), which are used to determine the dissociation constant (Kd), a key measure of affinity.

Affinity Determination

Affinity is determined from the kinetic data generated during the SPR experiment. The Biacore system provides real-time sensorgrams that display the binding interaction over time. From these sensorgrams, you can extract the following parameters:

• Ka (Association rate constant): The rate at which the antibody binds to the antigen.
• Kd (Dissociation rate constant): The rate at which the antibody dissociates from the antigen.
• KD (Dissociation constant): The equilibrium constant for the binding interaction, derived from the ratio of the dissociation rate constant (kd) to the association rate constant (ka).

The dissociation constant (Kd) is the key measure of affinity, where a lower Kd value indicates a higher affinity.

Affinity measurements by surface plasmon resonance (Yang et al., 2011).

Sensorgram Interpretation

Sensorgrams reflect the dynamic process of antibody-antigen binding, including the binding phase (antibody binding to antigen) and the dissociation phase (antibody dissociating from antigen). Sensorgrams can help to accurately measure antibody affinity. A typical sensorgram might exhibit:

• Steep increase in signal during association, corresponding to the antibody binding to the antigen.
• Plateau at maximum binding, indicating the saturation of the binding site.
• Gradual decrease during dissociation, as the antibody detaches from the antigen.

A well-behaved sensorgram should exhibit a smooth, monotonic dissociation phase, with a predictable kinetic profile that allows for accurate modeling of the affinity.

Applications of Biacore in Antibody Affinity Measurement

Screening and Characterization of Monoclonal Antibodies

High-throughput Screening of Antibody Libraries

Biacore systems offer a powerful platform for high-throughput screening (HTS) of large antibody libraries. By analyzing antibody-antigen interactions in real-time, Biacore facilitates the rapid identification of high-affinity candidates. This capability is particularly valuable for libraries derived from techniques such as phage display, B cell sorting, or other antibody discovery methods. By enabling efficient screening, Biacore helps streamline the selection of promising antibody candidates with optimal binding characteristics.

• Real-time binding data: The ability to observe binding events as they happen allows for the rapid selection of antibodies that bind to the target antigen with high specificity.
• Affinities and kinetics: Biacore provides not only the affinity (Kd) but also the association rate (ka) and dissociation rate (kd) of antibody-antigen interactions. This kinetic information is crucial for distinguishing antibodies with similar affinities but differing binding dynamics, such as slow off-rates that may be desirable for certain therapeutic applications.

Antibody Characterization and Optimization

Biacore is invaluable in characterizing antibodies throughout the drug development process. Once an antibody has been identified, detailed measurements of its binding affinity and kinetics are necessary to understand its potential as a therapeutic agent.

• Antibody binding specificity: Biacore can assess whether an antibody binds specifically to its target antigen or if there is cross-reactivity with other molecules, which is critical for ensuring the safety and efficacy of the drug.
• Determining the best candidates: By comparing binding profiles of multiple antibodies, Biacore helps prioritize candidates based on the optimal balance of affinity, specificity, and kinetics, which can influence therapeutic potency.

Evaluation of Antibody-Antigen Interactions in Complex Systems

One of the unique strengths of Biacore is its ability to analyze antibody affinity in complex biological matrices, such as serum, plasma, and cell lysates. The presence of multiple interfering components in these samples can complicate traditional assay methods, but Biacore's label-free nature and real-time measurements provide distinct advantages.

Measuring Affinity in Serum

In therapeutic antibody development, it is often necessary to evaluate the interaction of an antibody with its target in serum, where other proteins and molecules are present that could potentially interfere with the binding event. Biacore allows researchers to monitor the antibody-antigen interaction directly in the presence of such complex biological fluids, providing real-world data on how the antibody will perform in vivo.

• Competing interactions: Biacore can help identify how non-specific binding from serum proteins might influence the binding affinity of the antibody to its target antigen.
• Quantitative analysis: By using reference surfaces and appropriate buffers, researchers can subtract the background noise from the serum components, ensuring that the measured affinity reflects the true interaction between the antibody and its target.

Measuring Affinity in Cell Lysates

For many biologics, the target antigen is expressed on the surface of cells, or the interactions may occur in a more cellular context. Biacore's ability to assess antibody binding to targets on live cells or in cell lysates enables detailed analysis of these interactions, which are often more complex than in vitro interactions involving purified antigens.

• Cell surface antigens: Biacore enables the measurement of antibody binding to cell-surface antigens, such as those found in cancer immunotherapy targets. This is particularly useful in evaluating the functional activity of an antibody, especially when assessing cell-mediated immune responses.
• Real-time kinetics in lysates: When using cell lysates, Biacore can also provide kinetic data to better understand the dynamics of antibody-antigen binding in a more complex environment, which is closer to the actual conditions under which the therapeutic antibodies will function.

Analysis of Multivalent and Bispecific Antibody Interactions

In recent years, the development of bispecific antibodies (BsAbs) and other multivalent antibody formats has gained significant momentum. These engineered molecules are designed to bind to two or more targets simultaneously, offering enhanced therapeutic potential, particularly in cancer immunotherapy, autoimmune diseases, and other conditions requiring a multifaceted immune response.

However, the binding dynamics of multivalent antibodies can be far more complex than traditional monoclonal antibodies. Biacore provides the necessary analytical tools to characterize these complex interactions.

Bispecific Antibodies

Biacore is ideal for studying bispecific antibodies (BsAbs) because it allows for real-time measurement of the binding interactions of each antigen-binding site independently or in combination. This is particularly important for understanding:

• Kinetics of dual binding: Bispecific antibodies may exhibit cooperative binding (where binding to one antigen enhances the binding to the second) or competitive binding (where binding to one antigen prevents binding to the other). Biacore can help differentiate between these binding behaviors.
• Affinity balance: In the development of BsAbs, it is crucial to ensure that each binding site retains high affinity for its respective target while maintaining the integrity of the overall structure. Biacore's ability to measure both individual and overall binding affinities is invaluable in this regard.

Multivalent Antibodies

For multivalent antibodies or antibody fragments (such as diabodies or trivalent antibodies), the interaction can be more complex due to the presence of multiple binding sites. Biacore can provide insights into:

• Binding cooperativity: Multivalent interactions often involve binding to more than one epitope simultaneously. Biacore's kinetic analysis allows researchers to determine if the binding follows a cooperative model or if the individual sites behave independently.
• Optimizing valency: Understanding how the valency of an antibody impacts its binding affinity and overall therapeutic activity is essential for designing the most effective multivalent therapies.

Real-Time Monitoring of Antibody Therapeutics in Preclinical and Clinical Development

As antibody therapeutics move from discovery into preclinical and clinical development, it is essential to monitor their interactions with both the target antigen and other biomolecules within the body. Biacore's ability to measure binding affinity and kinetics in a range of conditions makes it an invaluable tool in these later stages of development.

Therapeutic Antibody Optimization

As therapeutic antibodies undergo engineering and optimization, such as through mutagenesis to improve their affinity or reduce immunogenicity, Biacore provides a platform to monitor these changes in real-time. By analyzing how small modifications to the antibody affect its binding kinetics and affinity, researchers can rapidly identify the most promising candidates for further development.

• Affinity maturation: In the process of affinity maturation, Biacore can be used to track the improvements in binding affinity over successive rounds of mutation and selection, helping to accelerate the development of high-affinity therapeutic antibodies.
• Therapeutic potency testing: Real-time affinity measurements are essential for testing the potency of an antibody in vivo. Biacore can simulate the conditions under which the antibody will function in patients, providing critical insights into its expected efficacy.

Batch Release Testing and Quality Control

During the manufacturing of therapeutic antibodies, Biacore is also used in quality control and batch release testing to ensure that the final product has consistent affinity and binding kinetics. This ensures that each batch of the therapeutic antibody performs as expected and meets regulatory standards.

Monitoring Antibody-Drug Conjugates (ADCs) and Immune Checkpoint Inhibitors

Antibody-drug conjugates (ADCs) combine antibodies with cytotoxic drugs, targeting them directly to cancer cells. Immune checkpoint inhibitors, such as those targeting PD-1/PD-L1 interactions, also rely on specific binding between antibodies and immune receptors. Biacore technology is used to:

• Measure ADC binding: Biacore can measure the affinity and kinetic properties of ADCs, helping developers to understand how the drug conjugate binds to its target and how the attachment of the cytotoxic payload affects its binding.
• Evaluate checkpoint inhibitor potency: Biacore can be used to evaluate the binding affinity of immune checkpoint inhibitors to their respective targets, such as PD-1 or CTLA-4, assessing their therapeutic potential.

Reference

1. Yang, Jiahui, et al. "5.1 Therapeutic Potential and Challenges of Targeting Receptor Tyrosine Kinase ROR1 with Monoclonal Antibodies in CLL." Clinical Lymphoma, Myeloma and Leukemia 11 (2011): S243.