Quantitative measurement of the target-mediated internalization kinetics of biopharmaceuticals

Pharm Res. 2015 Jan;32(1):286-99. doi: 10.1007/s11095-014-1462-8. Epub 2014 Sep 11.


Purpose: Measurement of internalization of biopharmaceuticals targeting cell surface proteins can greatly facilitate drug development. The objective of this study was to develop a reliable method for determination of internalization rate constant (kint) and to demonstrate its utility.

Methods: This method utilized confocal imaging to record the internalization kinetics of fluorescence-tagged biopharmaceuticals in live-cells and a quantitative image-analysis algorithm for kint determination. Kint was incorporated into a pharmacokinetic-pharmacodynamic (PK-PD) model for simulation of the drug PK profiles, target occupancy and the displacement of endogenous ligand.

Results: The method was highly sensitive, allowing kint determination in cells expressing as low as 5,000 receptors/cell, and was amenable to adherent and suspension cells. Its feasibility in a mixed cell population, such as whole blood, was also demonstrated. Accurate assessment of the kint was largely attributed to continuous monitoring of internalization in live cells, rapid confocal image acquisition and quantitative image-analysis algorithm. Translational PK-PD simulations demonstrated that kint is a major determinant of the drug PK profiles, target occupancy, and the displacement of endogenous ligand.

Conclusions: The developed method is robust for broad cell types. Reliable kint assessment can greatly expedite biopharmaceutical development by facilitating target evaluation, drug affinity goal setting, and clinical dose projection.

MeSH terms

  • Algorithms
  • Antibodies, Monoclonal / pharmacokinetics*
  • Antibodies, Monoclonal, Humanized
  • Biopharmaceutics / methods*
  • Carbocyanines / chemistry
  • Cell Line
  • Computer Simulation
  • Endocytosis*
  • Flow Cytometry
  • Fluorescent Dyes / chemistry
  • Humans
  • Models, Biological*
  • Molecular Imaging
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / genetics
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / metabolism*
  • Staining and Labeling


  • Alexa Fluor 647
  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Carbocyanines
  • Fluorescent Dyes
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor
  • mavrilimumab