Plasma Protein Binding Assays

protein-binding-red-device

Only unbound circulating drugs have access to targets and excretion pathways, therefore the binding of drug candidates to plasma proteins plays a significant role in efficacy, drug distribution, and establishing safety margins. For these reasons, pharmaceutical companies and regulatory agencies are interested in determining the extent of drug binding to plasma and/or tissue proteins early in the drug development process.

MicroConstants will measure the binding of test articles to plasma or tissue proteins using the following sequence:

  • Develop a specific and sensitive quantitative method
  • Validate the analytical assay in accordance with FDA guidelines (if desired)
  • Conduct a pilot study to determine the optimum methodology (equilibrium dialysis, ultrafiltration, or ultracentrifugation methods)
  • Perform the incubation, analysis, and calculations

Methods for Measuring Drug-Protein Binding

MicroConstants employs the following methods to determine unbound drug concentration in plasma, serum, or tissue:

  • Equilibrium dialysis
  • Ultrafiltration
  • Ultracentrifugation

The plasma or tissue sample is first spiked with the test article at one or more concentrations and then incubated with the appropriate matrix for a prescribed period of time; however, samples from clinical or toxicology studies can be used instead. The unbound concentration is then determined using one of the above mentioned assays followed by LC/MS/MS analysis. Equilibrium dialysis can be performed with the RED device or a Spectrum 20. Several concentrations can be used to calculate a dissociation constant, Kd.

Species and Isolated Proteins Available

We routinely measure and compare protein binding in plasma or serum from the following species:

  • Mouse, rat, rabbit, dog, monkey, and human
  • Hamster, guinea pig, and mini pig can be used if commercially available

Protein binding can also be assessed for individual proteins such as:

  • Human serum albumin
  • Human α1-acid glycoprotein
  • Human sex hormone binding protein

Protein Binding Study Options

Depending on your study needs, we will perform:

  • Submission-Quality Studies - exhaustive studies to generate protein binding data suitable for regulatory submissions
  • Screening Assays - assays to filter lead compounds and estimate protein binding values early in the discovery process

Submission-Quality Studies
A pilot study is conducted to determine which methodology (equilibrium dialysis, ultrafiltration, or ultracentrifugation) is most appropriate for the measurement of the unbound test article. During this study we will assess stability in plasma at 37ºC, non-specific binding to the apparatus, and the estimated protein binding ratio. The results from these assessments will allow us to select the method best suited to provide robust results for the test article.

During the pilot study, we may find it necessary to stabilize the compound with protease inhibitors, or to determine non-specific drug-protein binding for each concentration so we can normalize the binding ratio. Our extensive experience with protein binding studies enables us to handle compounds with limitations of stability and/or solubility.

Screening Assays
The compounds are spiked into plasma or serum and dialyzed overnight using the RED device. Following the incubation, the recovery samples, donor sides, and receiver sides are analyzed by time-of-flight mass spectrometry using a generic method. We then calculate the binding values and percent recovery from the relative peak heights.

When compounds with low recovery are encountered, we notify the sponsor and give them the option of investigating non-specific binding and stability in plasma.

Deliverables

An Excel spreadsheet with the drug-protein binding data will be submitted immediately after completion of the experiment. If desired, a formal report complete with the Abstract, Experimental, Results, and Conclusions can be provided as well. The formal report is suitable for inclusion in regulatory submissions.