Real-time, label-free protein binding analysis has long been considered challenging, especially for new or inexperienced users. However, the development of intuitive modern systems for performing techniques such as biolayer interferometry (BLI) and surface plasmon resonance (SPR) has altered this perception, leading to label-free detection methods being widely adopted in the past few decades. Nowhere is this more apparent than within the biopharmaceutical industry, where SPR has become the gold standard for determining accurate binding kinetics and affinity values. One of the main roadblocks to proper implementation of SPR experiments is optimization of the analyte concentration series for use in an assay. To address this, technology is now available to create a gradient injection of up to four-orders of magnitude from a single analyte concentration, significantly reducing the amount of time spent on assay development and assay performance compared to standard SPR methods.
SPR is an optical biosensing technique that measures changes in refractive index. It involves immobilizing a ligand on the sensor surface, before introducing the analyte into the system and allowing it to pass through the flow cell. As the analyte associates with the ligand, a corresponding increase in the response signal is seen. The analyte solution is then replaced with assay buffer to allow for dissociation, and the sensor is regenerated.
Determining the kinetics and affinity of the ligand-analyte interaction has traditionally involved performing multiple iterations of this process with different analyte starting concentrations. This allows for calculating the rate constants of association (ka) and dissociation (kd) and the equilibrium dissociation constant (KD), which are important criteria for lead selection. For example, a biologics discovery lab may have two lead candidates with similar affinity (KD) for the target of interest, but their differences in ka and kd can be used to estimate which biomolecule will be more useful in vivo. An example of multi-cycle binding data is shown in Figure 1.
Figure 1. Traditional multi-cycle kinetic analysis of analyte binding to an immobilized ligand with seven different concentrations of analyte.
A major drawback of traditional, multi-cycle kinetic analysis is that it adds preparation and analysis time to experimental workflows, increasing the potential for pipetting errors and evaporation. This can cause the analyte concentration to be incorrectly estimated, affecting the accuracy of ka, and subsequently the accuracy of KD. As a consequence, the wrong therapeutic could be progressed for further development at the expense of a more favorable candidate.
Gradient injection is a superior alternative to multi-cycle kinetic analysis that lets users determine binding kinetics and affinity values from a single analyte concentration. Based on the Taylor dispersion theory, which describes the analyte concentration in the flow cell over time as a function of the analyte diffusion coefficient, the flow rate, and dispersion line geometry, it produces a sigmoidal concentration gradient such that the full range of analyte concentrations presented to the sensor surface is incorporated into the analysis.
The gradient injection technology known as OneStep® represents a unique feature of the recently released Octet® SF3, Sartorius’ fully automated SPR system. OneStep® is capable of creating a gradient injection of up to four-orders of magnitude, subsequently using system-given constants to generate experimental results. This translates to savings in terms of reagents and plate space, as well as frees up researchers’ time to be spent on other tasks. An example of OneStep® binding data is shown in Figure 2.
Figure 2. OneStep® binding data for an analyte concentration gradient of 0–400 nM (teal) overlaid by a 1:1 kinetic model curve (red).
SPR with gradient injection technology has utility for many different applications, not least within the biopharmaceutical industry. These include ranking advanced hits for lead selection, characterizing high-affinity molecular interactions, and performing cell line development. As new classes of biologics with complex binding mechanisms are discovered, being able to efficiently analyze how these molecules interact with their targets will be key to bringing new drugs to market, faster.
Sartorius offers an extensive selection of products and resources for label-free detection, including the recently released Octet® SF3 system with OneStep® gradient injection. To learn more, visit sartorius.com