Protein Binding

Methods

• Method for Plasma Protein Binding

  • Test System: Pooled plasm from each species or specific plasma protein solutions (e.g., human serum albumin, α₁-acid glycoprotein, gamma-globulin)
  • Concentrations are selected to bracket known clinical exposures (0.1 – 10X C_max), unless limited by solubility.

  Equilibrium Dialysis

  Equilibrium dialysis is performed using a High Throughput Dialysis apparatus (HTDialysis LLC, Gales Ferry, CT).  A fortified matrix (either plasma or isolated plasma protein) is added to the donor chamber while Dulbecco’s Phosphate Buffered Saline (DPBS) is added to the receiver chamber of each well. Plates are then sealed with a gas-permeable membrane and incubated at 37°C in 5% CO₂ for the designated time (see below). After incubation, samples from each chamber are analyzed using liquid chromatography-mass spectrometry (LC-MS).

  • Stability in matrix: The test article is incubated in plasma and in DPBS at a single concentration of up to five time points.
  • Time to equilibrium: The test article is added to plasma at a single concentration and dialyzed against DPBS for up to five time points.
  • Protein binding concentration dependence: The test article is added to plasma at three concentrations and dialyzed against DPBS for the time required for equilibrium. 
  • A positive control such as warfarin is tested in parallel equilibrium dialysis for ≥5 hours.

  Ultrafiltration

  Fortified plasma will be added to the sample reservoir portion of an ultrafiltration device with a molecular weight cutoff of 30,000 Da. Samples will be centrifuged at 37°C and 2,000 × g for 15 minutes (or other appropriate conditions). After centrifugation, the ultrafiltrate will be analyzed by LC-MS for test article and content compared to initial concentrations. All protein binding determinations will be performed in triplicate. 

  • Nonspecific binding: The test article is added to control plasma ultrafiltrate (PUF)at two concentrations and centrifuged according to the ultrafiltration procedure. The dialysate will be analyzed to determine recovery of the test article and potential non-specific binding in the absence of plasma.
  • Protein binding concentration dependence: The test article is added to plasma at five concentrations and centrifuged according to the ultrafiltration procedure. The ultrafiltrate will be analyzed for test article.

  Ultracentrifugation

  Fortified plasma will be added to the ultracentrifuge tubes and centrifuged at 37ºC at 223,000 × g for 4 hours (or other appropriate conditions) to achieve separation of plasma ultracentrifugate from plasma protein. After centrifugation, the ultracentrifugate will be analyzed by LC-MS and compared to initial concentrations. 

  • Nonspecific binding: Test article is added to control human plasma and plasma ultrafiltrate (PUF) at two concentrations and incubated in ultracentrifuge tubes for 4 hours. The matrix will be analyzed to determine recovery of test article and potential non-specific binding in the absence of plasma.
  • Protein binding concentration dependence: Test article is added to plasma at five concentrations and centrifuged according to the ultracentrifugation procedure. The ultracentrifugate will be analyzed for test article.

• Method for Blood-to-Plasma Partitioning

  BPP studies are used to determine the in vitro blood-to-plasma partitioning of a test article in blood from various species, including human.

  • Test system: Blood obtained from each animal species may be pooled from at least three donors. Human blood obtained from at least three volunteers will not be pooled.
  • Hematocrit values are determined for the pooled animal blood samples and the individual human blood samples.
  • Blood is stored at 2-8°C and used as soon as possible within 1 week of receipt. 

  Test article is added to blood and initial aliquots are taken. Fortified blood is incubated at 37°C as indicated. Following incubation, aliquots are removed for analysis. Remaining blood is centrifuged to obtain plasma; aliquots are collected for analysis and compared to initial aliquots.

  • Stability in matrix: Test article is incubated in blood at a single concentration for up to four time points.
  • Time to equilibrium: Test article is added to blood at a single concentration for up to four time points.
  • Protein binding concentration dependence: Test article is added to blood at up to 5 concentrations and incubated to the equilibrium time point.

  For radiolabeled test articles, blood aliquots are solubilized prior to liquid scintillation counting (LSC). For non-radiolabeled test articles, blood aliquots are lysed prior to extraction and analysis using liquid chromatography-mass spectrometry (LC-MS).

• Method for Microsomal Protein Binding

  An assessment of the potential for a test article to bind to human liver microsomal protein in vitro allows for correction of unbound fraction in assays using these systems.

  Equilibrium dialysis is performed in triplicates using a High Throughput Dialysis apparatus (HTDialysis LLC, Gales Ferry, CT).  Fortified microsomes are added to the donor chamber while an assay buffer is added to the receiver chamber of each well. Plates are then sealed with a gas-permeable membrane and incubated at 37°C in 5% CO₂ for 5 hours. After incubation, samples from each chamber are analyzed using liquid chromatography-mass spectrometry (LC-MS).

  • Protein binding: Human liver microsomes are diluted to three concentrations (0.05, 0.1, 0.5 mg/mL) in phosphate buffer. The test article is then added to microsomes at three concentrations for a total of 9 samples. Fortified matrix samples are transferred to HTD donor chambers in triplicate and dialyzed against phosphate for 5 hours.
  • Stability in microsomes: Fortified matrix samples from above are incubated at 37°C in 5% CO₂ for 0 and 5 hours.