Nobel laureate Paul Ehrlich famously coined an expression: drugs will not act unless they are bound.¹⁻² With this accepted truth, many drug developers focused on generating drug candidates with strong binding affinity. This classical approach was challenged by numerous clinical failures which gave rise to more detailed models including binding times or residency times, and ultimately to the evolution of advanced scientific approaches and methodologies for the mechanistic characterization of even the simplest of binding interactions. In the case of biological molecules, binding complexity is magnified with the introduction of allostery, post-translational modifications, degradation, and higher-order structure formation.
Surface Plasmon Resonance (SPR) is a widely used, highly sensitive technique for assessing molecular binding events in real-time. From small molecules to large proteins, or even viruses, the technique has a number of applications in the Biopharmaceutical industry, which include, but are not limited to:
- Discovery; candidate screening and selection
- Drug development; characterization, comparability, and biosimilarity
- Commercial; batch release and stability
A typical SPR setup is shown in Figure 1. SPR measures a change in refractive index at the gold sensor surface. One of the binding partners under study is immobilized on the sensor surface and the other binding partner is passed over it using microfluidics. When polarized light is directed through a glass prism to the bottom of the sensor surface, total internal reflection conditions cause electrons in the gold to absorb light at a specific angle, known as the resonance angle, and this results in an intensity minimum at the detector. As the binding partners interact, the angle of the intensity minimum shifts as a function of time. This response is presented in the form of a sensorgram.
A typical SPR assay involves five steps as shown in Figure 2.
- Preparation of suitable buffers, reagents, and samples.
- Immobilization of one of the molecules involved in the binding study to the sensor surface.
- Binding of the analyte as it is passed over the surface containing the immobilized binding partner.
- Regeneration of the sensor to restore the surface back to its unbound state.
- Data Analysis to determine kon rates, koff rates, and therefore association or dissociation constants, concentrations or other parameters.
SPR provides a highly specific, highly sensitive, label-free, low volume means of determining real-time association/dissociation constants between molecules. The absence of a label lends an advantage in that the risk of molecular alterations or introduction of steric interference is reduced. Real-time measurements provide critical kinetic data which often correlates with function and potency and allows for kinetic level evaluations to be performed. This provides a strong alternative to other techniques such as ELISA, or FRET. In addition, with a strong understanding of protein binding, calibration-free concentration analysis is also possible in more complex matrices than would otherwise be possible by alternative techniques.
But what are the drawbacks? With great power, comes great responsibility.
In practice, SPR technology can be expensive to establish and maintain. While recent years have introduced competition within the SPR vendor market, precision technology remains expensive. Readily available alternatives may provide orthogonal data sets and still achieve the same outcome. From a maintenance perspective, SPR systems themselves are generally robust, but do require daily in-house care, and specialized external maintenance on a routine basis.
What about ease of use? This is the elephant in the room. A lot of effort has been made to make system interfaces user friendly, and to standardize workflows; but despite all this, the complexity of the science remains. Established test methodologies require well-trained, and qualified personnel to perform the work. Development of methodologies, especially those intended to support regulatory applications requires careful thought, and specialized expertise. The application of these methodologies to support various study designs intended to evaluate stability, comparability, or biosimilarity also requires specialized, and experienced scientists to perform and evaluate the data carefully.
In the end, whether it be characterization, biosimilarity, or batch release, SPR is a robust, but complex analytical tool that provides paramount value to the development and understanding biopharmaceutical products. With a well-trained, experienced team the full power of SPR can be realized.
¹ Yan, W. (2015) "Binding time--not just affinity--gains stature in drug design." Nature Medicine, 21:545.
² Bosch, F. and Rosich, L. (2008) "The Contributions of Paul Ehrlich to Pharmacology: A Tribute on the Occasion of the Centenary of His Nobel Prize." Pharmacology, 82(3): 171-179