Assessment of serum protein binding to predict non-specific uptake in vivo

Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Translational - Applied Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs


Method keywords
  • Serum protein binding
  • hepatobiliary clearance
  • size-exclusion chromatography
Scientific area keywords
  • in vivo biodistribution
  • non-invasive imaging
  • pharmacokinetics
Method description

For therapeutic or reporter molecules to be effective for therapy or imaging applications, proper accumulation of the compounds in the tissue of interest is required, with minimal accumulation in undesired organs to avoid toxic side-effects and increase bioavailability. After initial in vitro screening on functionality and potency, a panel of preselected analogues are often evaluated in vivo for their pharmacokinetic profile to select a lead compound. This is most often assessed in vivo following administration of the compound using chromatography techniques or ELISA on collected tissues/organs, or non-invasive imaging. We propose to screen the compounds beforehand for non-specific protein binding in order to predict undesired background accumulation, in particular in the liver. As such, the number of compounds that need to be tested in vivo can be limited to the most promising ones.

Lab equipment

HPLC Fluorescent- or radiolabeled drug-analogue Serum

Method status
  • History of use

Pros, cons & Future potential


By screening beforehand a panel of drugs on their serum protein binding, the number of preselected molecules that need to be evaluated in vivo can be reduced as these will exhibit in vivo non-specific binding, in particular liver accumulation. Depending on the application, this can lead to undesired toxic side-effects and decrease the bioavailability of the drug.


To differentiate the signal of the drug from the signal of the serum proteins, the drug needs to be labeled either with a fluorophore or a radioisotope. One should be aware that the modification of a drug can also impact the biodistribution profile. In particular, hydrophobic fluorescent dyes are known to exhibit non-specific protein binding. Moreover, one should be reminded that the in vitro assay is a static assay that does not necessarily reflect the dynamic process happening in vivo and that metabolization, clearance, compartimentalization etc is not taken into account. Moreover, this assay does not predict in vivo efficacy of the compounds.


This method could be adapted to screen the binding of drugs to other types of proteins that can predict non-specific accumulation in certain undesired organs/tissues in the body.

Future & Other applications

This method could be used to screen additional types of drugs (small molecules, peptides, large antibodies) for their non-specific serum protein binding and as such predict their in vivo behavior. This will limit the number of compounds tested in vivo.

References, associated documents and other information

  • P. Debie, C. M. Huygen, B. De Sloovere, D.M. van Willigen, Ł Mateusiak, N. B. Declerck, J. Bridoux, J. Puttemans, N. Devoogdt, F. W. B. van Leeuwen, S. Hernot. Design and preclinical evaluation of a single-label bimodal nanobody tracer for image-guided surgery. Biomolecules (2021) 11, 360. IF2020 4.879
  • D.A. Smith, L. Di & E.H. Kerns. The effect of plasma protein binding on in vivo efficacy: misconceptions in drug discovery. Nature Reviews Drug Discovery (2010) 9, 929–939
  • Additional publications upcoming
Associated documents

Contact person

Sophie Hernot


Vrije Universiteit Brussel (VUB)