Skip to main content

Surface Plasmon Resonance (SPR) for characterizing the affinity and kinetics for monoclonal antibodies

 Surface Plasmon Resonance (SPR) is a powerful technique for characterizing the affinity and kinetics of interactions between biomolecules, such as nivolumab (a monoclonal antibody) and its target, PD-1. SPR provides real-time, label-free measurements of binding interactions, allowing determination of the binding kinetics (association and dissociation rates) and affinity constant (Kd).

Here’s a step-by-step procedure for SPR to characterize the binding affinity of nivolumab to PD-1:

1. Materials and Reagents

  • Ligand: Recombinant human PD-1 protein (ligand, which will be immobilized on the sensor chip)
  • Analyte: Nivolumab (as the binding analyte flowing over the sensor chip)
  • Running buffer: Typically PBS or HEPES-buffered saline (HBS) with added surfactant (e.g., 0.05% Tween-20) to reduce non-specific binding
  • Regeneration buffer: A solution to dissociate nivolumab from PD-1 without denaturing the immobilized PD-1 (often an acidic buffer, e.g., glycine-HCl at pH 2.5–3.5, or NaCl at high concentrations)

2. Setting Up the SPR Instrument

  • Calibrate the SPR system according to the manufacturer’s instructions.
  • Prime the fluidics system with the running buffer to ensure consistent flow and stability of baseline signals.

3. Sensor Chip Selection and Preparation

  • Sensor Chip Selection: Use a sensor chip that is compatible with protein immobilization, such as a CM5 chip (carboxymethylated dextran) commonly used for protein-protein interactions.
  • Ligand Immobilization (PD-1): Use either amine coupling, where PD-1 is covalently attached to the chip, or capture methods if the ligand needs to be regenerated frequently.
    • Amine Coupling:
      1. Activate the carboxyl groups on the chip surface by injecting a mixture of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide).
      2. Dilute PD-1 protein in an appropriate buffer (e.g., sodium acetate buffer, pH 4.5–5.5).
      3. Inject PD-1 onto the activated surface at a controlled concentration to achieve the desired immobilization level.
      4. Deactivate the surface with ethanolamine to block any remaining active sites and stabilize the ligand attachment.
  • Aim for immobilizing a low level of PD-1 on the chip to minimize mass transport effects and allow accurate kinetic measurements.

4. Running the Experiment

  • Baseline Stabilization: Allow the running buffer to flow over the sensor surface until a stable baseline is achieved.
  • Binding Assay:
    • Prepare nivolumab dilutions across a concentration range (e.g., from low nanomolar to micromolar) to characterize binding kinetics accurately.
    • Inject each concentration sequentially over the sensor surface with immobilized PD-1, allowing nivolumab to bind to PD-1.
    • Monitor real-time changes in SPR signal (in resonance units, RU) as nivolumab associates with PD-1 on the chip.
  • Dissociation Phase:
    • Stop the injection of nivolumab and allow the running buffer to flow over the chip. This phase records the dissociation kinetics as nivolumab gradually detaches from PD-1.

5. Regeneration of the Sensor Surface

  • After each cycle, inject the regeneration buffer to remove bound nivolumab from PD-1 without damaging the immobilized PD-1.
  • After regeneration, flush with running buffer to return to baseline and prepare for the next sample injection.

6. Data Analysis and Interpretation

  • Association Rate Constant (ka): Measure the rate of nivolumab binding to PD-1. The association rate is derived from the slope of the association phase in the sensorgram.

  • Dissociation Rate Constant (kd): Measure the rate at which nivolumab dissociates from PD-1. The dissociation rate is derived from the slope of the dissociation phase in the sensorgram.

  • Affinity Constant (Kd): Calculate the affinity constant using the formula:

    Kd=kdkaK_d = \frac{k_d}{k_a}

  • Fit the binding curves with a 1:1 binding model (if appropriate) or other models if the interaction displays complexity.

Key Considerations

  • Temperature Control: Conduct the experiment at physiological temperature (e.g., 25–37°C) for accurate kinetic data.
  • Concentration Range: Ensure that the range of nivolumab concentrations spans both above and below the expected Kd to obtain robust data.
  • Mass Transport Limitation: Keep the ligand density low on the sensor chip to reduce mass transport effects, which can interfere with accurate kinetic measurements.

Summary

SPR provides precise real-time measurements of nivolumab’s affinity to PD-1 by determining both association and dissociation rates, enabling the calculation of the affinity constant. This technique is essential for developing and confirming nivolumab’s strong target specificity and favorable binding kinetics for therapeutic efficacy.

Popular posts from this blog

Ago2 Immunoprecipitation for RISC-siRNA Quantitation

 Ago2 (Argonaute 2) immunoprecipitation (IP) is a technique used to isolate RNA-induced silencing complexes (RISC) from cell lysates. This method allows for the specific enrichment of active RISC complexes bound to small interfering RNA (siRNA) or microRNA (miRNA) within cells. By isolating these complexes, researchers can then quantify the siRNA associated with Ago2, which is an essential step in determining the efficacy of RISC loading and siRNA activity. Here’s a detailed overview of how Ago2 immunoprecipitation is performed for RISC-siRNA quantitation: Steps in Ago2 Immunoprecipitation for RISC-siRNA Quantitation Cell Lysis and Preparation of Lysate : Sample Preparation : Collect cells that have been treated with siRNA, then wash them with cold phosphate-buffered saline (PBS) to remove extracellular contaminants. Lysis : Lyse the cells in a gentle, RNA-preserving lysis buffer that typically includes detergents (e.g., NP-40 or Triton X-100), protease inhibitors, and RNase inhibi...
Read more

ICH E3 Structure and content of clinical study reports (CPMP/ICH/137/95)

 The ICH E3 guideline, titled "Structure and Content of Clinical Study Reports," with the reference number CPMP/ICH/137/95, provides recommendations and a standardized framework for the structure and content of clinical study reports (CSRs). CSRs are essential documents that summarize the results and findings of clinical trials conducted during the drug development process. Here's an elaboration of ICH E3: 1. Purpose: The primary purpose of ICH E3 is to provide guidance on the organization, content, and format of CSRs to ensure consistency and clarity in reporting clinical trial data. It aims to facilitate the evaluation of the safety and efficacy of investigational drugs by regulatory authorities. 2. Applicability: ICH E3 is applicable to CSRs for all phases of clinical trials, including Phase I, II, III, and post-marketing studies. 3. Structure of the CSR: The guideline outlines a standardized structure for the CSR, which typically includes the following sections: Title...
Read more

ICH Q5D Derivation and characterisation of cell substrates used for production of biotechnological/biological products (CPMP/ICH/294/95)

The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) provides guidelines to ensure the quality, safety, and efficacy of pharmaceutical products. ICH Q5D, as outlined in document CPMP/ICH/294/95, addresses the derivation and characterization of cell substrates used for the production of biotechnological and biological products. Below is a detailed elaboration of ICH Q5D: 1. Purpose of ICH Q5D: ICH Q5D provides guidelines for the establishment of cell substrates used in the production of biotechnological and biological products. The primary goal is to ensure the quality, safety, and consistency of cell substrates to minimize potential risks associated with the final product. 2. Cell Substrate Characterization: The guideline emphasizes the importance of thorough characterization of the cell substrate. This includes the origin of the cells, their history, and any relevant genetic information. Detailed documentation of the cell line...
Read more

Safety Concerns for AAV Gene Therapy

 Adeno-associated virus (AAV) gene therapies have shown significant therapeutic promise, but they also carry risks, and toxicity signals are a primary safety concern. While generally well-tolerated, AAV-based therapies can trigger adverse effects ranging from immune-related responses to cellular toxicities, especially at higher doses. Here’s an overview of the key toxicity signals associated with AAV gene therapy, along with potential mechanisms and mitigation strategies: 1. Liver Toxicity Signal : Hepatotoxicity is one of the most common toxicity signals with AAV gene therapy, especially with high vector doses or in patients with pre-existing liver disease. Mechanism : AAV vectors, often targeting the liver, can cause liver inflammation due to: Immune responses to AAV capsids. Overexpression of the therapeutic transgene, leading to cellular stress. Clinical Signs : Elevated liver enzymes (ALT, AST) are common indicators of hepatotoxicity. Mitigation : Strategies include using immu...
Read more

ICH Topic Q5E Comparability of biotechnological/biological products (CPMP/ICH/5721/03)

 ICH Topic Q5E, as outlined in document CPMP/ICH/5721/03, deals with the comparability of biotechnological and biological products. This guideline provides a structured framework for assessing and ensuring the comparability of different product versions, including changes during development, manufacturing, or post-approval phases. The goal is to demonstrate that changes made to a product do not adversely affect its quality, safety, or efficacy. Here's an elaboration of ICH Q5E: 1. Purpose of ICH Q5E: The primary purpose of ICH Q5E is to provide guidance on how to demonstrate the comparability of biotechnological and biological products, especially when changes are made to the manufacturing process or product characteristics. Comparability studies are crucial for ensuring the consistent quality and safety of these products. 2. Types of Changes Covered: ICH Q5E covers a wide range of changes, including modifications to the manufacturing process, changes in the manufacturing site, alt...
Read more