This article from Issue 10 of the Analytix Reporter, produced by Merck, provides a comparative study between the Supel™ BioSPME 96-Pin device and the rapid equilibrium dialysis technique.
An important aspect of drug discovery is understanding the interaction of the drug candidate with plasma proteins and lipids. The binding of drugs to the proteins and lipids is referred to as the plasma protein binding (PPB or Fb). The molecular attributes of a drug can provide useful insights into its number of interactions with the protein. In general, organic acids have a single binding site with albumin, whereas, organic bases have multiple bindings sites associated with glycoproteins. In addition to albumin, other proteins commonly associated with drug binding are alpha1-acid glycoprotein (AAG) and lipoproteins, such as very high-density lipoprotein (VHDL) and low-density lipoprotein (LDL). When measuring the pharmacologic potency of a drug, it is the free fraction or unbound fraction (Fu) of a drug that is generally considered responsible for its activity as described by the free drug hypothesis.
Determining the protein binding properties of a drug is important to understand the amount of free drug available in the blood. So far, equilibrium membrane dialysis has been the traditional technique used to measure drug protein binding. The technique involves equilibration of a drug rich plasma sample with a drug-free buffer across a membrane. This allows the free drug to migrate across the membrane and prevents the protein bound drug from moving into the buffer. This equilibrium takes more than 24 hours to establish. Other techniques such as rapid equilibrium dialysis further reduce the workflow time from >24 hours to 6 hours by using specifically designed devices.
In this study, the Supel™ BioSPME 96-Pin device is used to measure drug protein binding. Supel™ BioSPME 96-Pin devices have been developed using solid phase microextraction, SPME, technology to extract free unbound analytes from biological fluids. These devices consist of a 96-pin plate, with the tips of pins coated with a thin layer of adsorbent particles. The patented binder within the coating allows the small analytes of interest to bind, while preventing macromolecules from binding. This enables a robust, selective, and non-exhaustive extraction of free analytes, having both qualitative and quantitative applications. The 96-pin configuration allows direct sampling from 96 well plates and is compatible with robotic liquid handling systems, providing a fully automated high-throughput methodology.
In this study, the Supel™ BioSPME 96-Pin device workflow is compared with a rapid equilibrium dialysis technique to measure drug protein binding. A series of compounds with molecular weights in the range of 230-750 Da and a hydrophobicity (LogP) range of 1.5-5 were utilized for comparison of protein binding values, and determined using each technique. In addition to measured values and accuracy, the study compared overall sample cleanliness and workflow time of each technique.
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Results & Discussion
The Supel™ BioSPME method determines the free concentration of analyte in plasma by comparing it with the extraction of the same analyte from buffer samples; where 100% of the analyte is considered to be free of protein binding. Supel™ BioSPME 96-pin devices were directly compared with a rapid equilibrium dialysis technique, as it is often considered the standard approach and workflow of choice for plasma protein binding determination.
The Supel™ BioSPME 96-Pin device technique has shown significant time savings for protein binding
determination when compared with the rapid equilibrium dialysis method.
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