Measurement of Free Versus Total Therapeutic Monoclonal Antibody in Pharmacokinetic Assessment is Modulated by Affinity, Incubation Time, and Bioanalytical Platform.
Decisions about efficacy and safety of therapeutic proteins (TP) designed to target soluble ligands are made in part by their ex vivo quantification. Ligand binding assays (LBAs) are critical tools in measuring serum TP levels in pharmacokinetic, toxicokinetic, and pharmacodynamic studies. This study evaluated the impact of reagent antibody affinities, assay incubation times, and analytical platform on free or total TP quantitation. An ELISA-based LBA that measures monoclonal anti-sclerostin antibody (TPx) was used as the model system. To determine whether the method measures free or total TPx, the effects of K on, K off, and K D were determined. An 8:1 molar ratio of sclerostin (Scl) to TPx compared to a 1:1 molar ratio produced by rabbit polyclonal antibodies to TPx was required to achieve IC50, a measure of TPx interference effectiveness, making it unclear whether the ELISA truly measured free TPx. Kinetic analysis revealed that Scl had a rapid dissociation rate (K off) from TPx and that capture and detection antibodies had significantly higher binding affinities (K D) to TPx. These kinetic limitations along with long ELISA incubation times lead to the higher molar ratios (8:1) required for achieving 50% inhibition of TPx. However, a microfluidic platform with the same reagent pairs required shorter incubations to achieve a lower Scl IC50 molar ratio (1:1). The findings from this study provide the bioanalytical community with a deeper understanding of how reagent and platform selection for LBAs can affect what a particular method measures, either free or total TP concentrations.
Department of Pharmacokinetic and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320, USA.|Regeneron Pharmaceuticals, 81 Columbia Turnpike, Rensselaer, New York, 12144, USA.
Jeffrey J
JJ
Talbot
Department of Pharmacokinetic and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320, USA.
Dominador
D
Calamba
Department of Pharmacokinetic and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320, USA.|Department of Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California, USA.
Melody
M
Pai
Department of Pharmacokinetic and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320, USA.
Mark
M
Ma
Department of Pharmacokinetic and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320, USA. theingi.thway@amgen.com.
Theingi M
TM
Thway
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood* / Q000493:pharmacokinetics*; D019485:Bone Morphogenetic Proteins / Q000097:blood* / Q000493:pharmacokinetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005819:Genetic Markers; D006801:Humans; D008024:Ligands; D051379:Mice; D011485:Protein Binding / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D008024:Ligands; C422903:SOST protein, human
affinity and kinetics; association and dissociation; free versus total; interference; ligand binding assay