Developing Protein Binding Assays Early in the Drug Development Process

Unbound circulating drugs have the best 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.

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Protein Binding Analysis: A 4-Step Sequence

Our scientists measure the binding of test articles to plasma or tissue proteins using the following sequence:

  1. Develop a specific and sensitive quantitative method.
  2. Validate the analytical assay in accordance with FDA guidelines (if desired).
  3. Conduct a pilot study to determine the optimum methodology (equilibrium dialysis, ultrafiltration, or ultracentrifugation methods).
  4. 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:

  • Serum albumin (multiple species commercially available)
  • Human α1-acid glycoprotein
  • Human sex hormone binding protein
  • Human gamma globulins
  • Low and high density lipoprotein
  • Human thyroxine binding globulin
  • Human prealbumin (aka transthyretin)

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.

Equipment and Software for Protein Binding Assays

MicroConstants is one of the largest bioanalytical LC/MS/MS laboratories on the West Coast of the United States. The instrumentation at our facilities is state-of-the-art and constantly upgrades equipment to remain at the forefront of the industry.

See Our Complete Equipment and Software List