4) Procedure

The following steps outline the detailed procedure for conducting Surface Plasmon Resonance (SPR) studies:

1. Step 1: Preparation of SPR Sensor Chip
   1. Choose the appropriate sensor chip based on the type of interaction being studied. For example, use a CM5 chip for protein-ligand interactions or a C1 chip for smaller molecule binding studies.
   2. Activate the sensor chip surface using an appropriate chemistry (e.g., NHS/EDC activation) to covalently immobilize the target protein or receptor.
   3. Immobilize the target protein or receptor on the chip surface by injecting it in a suitable buffer and monitoring the surface plasmon resonance signal during the immobilization process. Ensure that the immobilization level is within the optimal range for the SPR assay.
   4. Block any unreacted sites on the chip surface with an appropriate blocking reagent (e.g., ethanolamine) to prevent non-specific binding.
2. Step 2: Sample Preparation
   1. Prepare the ligand (e.g., small molecule, peptide) in appropriate concentrations for SPR analysis. Ensure that the ligand is dissolved in a suitable solvent and buffer to avoid interference with the SPR signal.
   2. For kinetic studies, prepare a series of ligand concentrations to be tested against the immobilized target, ensuring that the concentrations cover the expected binding range.
   3. Ensure that the ligand does not contain contaminants that could interfere with the interaction or cause non-specific binding to the sensor chip surface.
3. Step 3: Running the SPR Experiment
   1. Inject the prepared ligand solutions over the sensor chip surface, and measure the binding kinetics in real-time. Monitor the resonance signal (in response units, RU) as the ligand binds to the immobilized target.
   2. Record the association phase (when the ligand binds to the target) and the dissociation phase (when the ligand dissociates from the target) for each ligand concentration.
   3. Use appropriate flow rates (e.g., 10–30 µL/min) to ensure that the interactions are measured accurately without affecting the stability of the binding event.
   4. For kinetic analysis, inject multiple concentrations of the ligand to obtain a dose-response curve and assess the binding affinity.
4. Step 4: Data Analysis
   1. Analyze the SPR sensorgram to extract binding parameters such as association rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (Kd).
   2. Fit the sensorgram data using suitable software (e.g., BIAevaluation, Scrubber, or Origin) to obtain accurate kinetic parameters. For simple binding, a 1:1 Langmuir model is typically used to calculate these values.
   3. For more complex interactions, use advanced fitting models (e.g., bi-molecular interaction models) to assess multi-step binding mechanisms.
   4. Use control experiments (e.g., buffer injections) to subtract non-specific binding signals from the raw data and ensure accurate results.
5. Step 5: Interpretation of Results
   1. Interpret the SPR data to determine the binding affinity of the ligand for the target protein. A low Kd indicates a high-affinity interaction, which is desirable for drug candidates.
   2. Analyze the kinetic parameters to understand the binding mechanism. For example, a fast association rate (kon) and slow dissociation rate (koff) indicate strong binding, which may be favorable for drug efficacy.
   3. Compare the binding characteristics of different compounds to identify the most potent candidates for further development.
6. Step 6: Documentation and Reporting
   1. Document all experimental parameters, including sensor chip preparation, ligand concentrations, buffer conditions, flow rates, and SPR data analysis methods.
   2. Prepare an SPR Report that includes the binding affinity (Kd), association and dissociation rates (kon and koff), and other relevant kinetic parameters. Include the raw data, sensorgrams, and analysis results in the report.
   3. Ensure that all data is stored securely for future reference, regulatory compliance, and intellectual property purposes.

5) Abbreviations

• SPR: Surface Plasmon Resonance
• RU: Resonance Units
• kon: Association Rate Constant
• koff: Dissociation Rate Constant
• Kd: Dissociation Constant

6) Documents

The following documents should be maintained throughout the SPR process:

1. SPR Experimental Protocol
2. SPR Data and Sensorgrams
3. SPR Data Analysis Report
4. SPR Binding Affinity and Kinetic Parameters Report

7) Reference

References to regulatory guidelines and scientific literature that support this SOP:

• FDA Guidance for Industry on Drug Discovery and Molecular Interactions
• Scientific literature on SPR methodology in drug discovery

8) SOP Version

Version 1.0: Initial version of the SOP.