Plasma protein binding


The pharmacokinetic and pharmacodynamic properties of the compound are affected by its affinity to bind the abundant plasma proteins, like albumin, globulins and alpha-1-acid glycoproteins (basic compounds typically bind to alpha-1-acid glycoproteins and acidic componds typically bind to albumin). Binding to plasma proteins affects the compounds bioavailability and tissue distribution. The results form plasma protein binding should be taken into account when extrapolating the in vitro to in vivo kinetics and clearance. Reversibel high or low amount of compound bound to the plasma proteins is seldom itself a reason to discontinue the compounds development, see an excellent review on the topic (Smith DA, Di L, Kerns EH. The effect of plasma protein binding on in vivo efficacy: misconceptions in drug discovery. Nat Rev Drug Discov. 9(12):929-39, 2010.

Protein binding is typically analyzed in one of three methods: rapid equilibrium dialysis (RED), ultrafiltration, or ultracentrifugation, followed by LC-MS/MS quantification.

  • RED method uses an equilibrium dialysis membrane device (low MW cut-off, so proteins can not cross the membrane); plasma on one side of the membrane and buffer, like PBS, on the other side. Tested compound is added to the plasma side and after a time period, like 4 hrs, the both compartments are diluted with equal volume of the other solution (plasma with equal volume of PBS and PBS with equal volume of blank plasma, respectively; this is to equalize the analyzed matrices). Equal volumes are sampled on both solutions, proteins are precipitated by acetonitrile and supernatants are analysed with LC-MS/MS. The results are expressed simply as a ratio between the samples taken from both sides of the membrane.
  • Ultrafiltration is based on first incubating the compound in plasma (typically shorter period of time, like 30 min), followed by filtering the plasma-compound mixture through a low MW cut off filter, which proteins can not cross, by centrifugation. Fraction of the compound unbound to proteins is quantitated from the liquid that crossed the filtration membrane. Ultrafiltration is a bit more tedious method, since a set of standards will have to be analyzed the same way as well. The analysis method is alike LC-MS/MS.
  • Ultracentrifugation is the third, and typically less used method and most tedious method for detecting compound’s protein binding. In ultracentrifugation, very high speed ultracentrifuges are used, which separated the non-bound compound from the protein bound compound.

Our expertise in plasma protein binding

We offer analysis of binding to plasma proteins with two methods, namely ultrafiltration (centrifugation) and rapid equilibrium dialysis.

Plasma protein binding analysis by rapid equilibrium dialysis (RED)

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Plasma protein binding analysis by ultrafiltration

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