EScalate Assay

Accurate determination of plasma protein binding (PPB) has been a challenge for many years as a decent fraction of compounds from different classes such as:

  • Peptides and peptidometics
  • Oligonucleic acids
  • Small molecules

tends to fail in conventional assays. The inability to pass dialysis membranes, adhesion or stickiness, instability and buffer insolubility are the key reasons for flawed results. Moreover, traditional methods exhibit a high variation of experimental results for very strong serum protein binders with an unbound fraction (fu) above 98%.

3BP developed the EScalate® Equilibrium Shift Assay which is broadly applicable and provides a remarkable solution for PPB analysis without the limitations described above.

The EScalate Equilibrium Shift Assay can be considered the first assay technology by which the free fraction in plasma of moderate to ultra-strong binders can be reliably determined. Key features of the assay are:

  1. Excellent data accuracy, also for very strong serum binders
  2. Sophisticated data quality control
  3. Ability to cope with non-specific interactions of the test compounds (“stickiness”)
  4. Rapid equilibration enabling the analysis compounds not yet optimized for stability
  5. Highly selective MS detection excluding distortion caused by metabolites

Typical research questions

The EScalate assay can be employed to answer the following experimental questions:

  • What is the free fraction (fu) of my experimental compound in plasma?
  • How does the fu of my compound compare to that of a specific reference compound?
  • Is the free fraction in plasma consistent across a set of relevant experimental models (mouse, rat, human, cynomolgus, mini-pig, etc.) ?
  • Is plasma protein binding of my compound driven by HSA (human serum albumin) or AGP (alpha 1 acid glycoprotein) ?
  • Which chemical derivative of my compound leads to the desired level of plasma protein binding and is this finding consistent across all investigated species?

The assay principle

The EScalate assay comprises a microtiter plate-based two-dimensional dilution matrix of blood plasma and HSA-coated beads plus a set of appropriate controls. The impact of increasing plasma concentration on the binding equilibrium of a test compound to HSA-coated beads is determined experimentally. Samples are subsequently quantified by LC-MS. From the acquired data set, the free fraction (fu) of the test compound in plasma is calculated. Results can be determined for any species for which plasma is available. For details please refer to our publications on the EScalate assay.

Sample Processing

EScalate samples are processed by 3B Pharmaceuticals in Berlin, Germany. Depending on the number of compounds and species to be analyzed, results will typically be available four to six weeks after receipt of the test compounds at 3B Pharmaceuticals’ facilities.

EScalate is a registered trademark of 3B Pharmaceuticals GmbH.