Technical Note: Free, Bound, and Total Drug Estimation of monoclonal antibody drugs
Understanding the pharmacokinetic and pharmacodynamic behavior of a given therapeutic drug is an essential element of understanding its effectiveness and safety, as well as identifying the proper dosage, combinations and distribution.
All pre-clinical and clinical studies include the measurement of serum drug concentration and anti-drug antibodies, both in animals and in patients, at different points after drug administration. The result is an important indicator of the drug’s pharmacokinetic and immunogenic properties and is pertinently relevant to dosing recommendations while developing the drug, and even while using in clinical therapy.
When the mAb and the target both circulate in vivo, various molecular species of mAb and target co-exist in a dynamic equilibrium including free drug, free circulating target, total drug and the total target-drug complex.
It has long been acknowledged that data on different drug and target species (e.g., free vs total levels of drug target as two possible biomarkers) may satisfy different needs in the research and development of new drugs.
Depending on the information required for decision-making, the data required and hence the selection of assay (free, total or both) may differ at each phase of drug development.
Unbound (free) drug concentrations in plasma are readily measurable and are often used as surrogates for the drug concentrations at the site of pharmacologic action in pharmacokinetic-pharmacodynamic analysis and clinical dose projection in drug discovery.
Furthermore, for permeable compounds at pharmacokinetic steady state, the free drug concentration in tissue is likely a close approximation of that in plasma; however, several factors can create and maintain disequilibrium between the free drug concentration in plasma and tissue, leading to free drug concentration asymmetry.
Free drug levels reflect the availability of the active drug, while the total or bound drug complex is of importance when checking for efficacy or dynamic interactions of the drug, and for other PK/PD assessments. These factors include drug uptake and extrusion mechanisms involving the uptake and efflux drug transporters, intracellular biotransformation of prodrugs, membrane receptor–mediated uptake of antibody-drug conjugates, pH gradients, unique distribution properties (covalent binders, nanoparticles), and local drug delivery (e.g., inhalation).
Thus, it is important to understand the information needed at different stages of drug development.
| FREE DRUG ASSAY | BOUND DRUG ASSAY | TOTAL DRUG ASSAY |
|---|---|---|
| These antibodies inhibit binding of the drug to its target | These antibodies recognize only the bound drug-target complex, and do not bind to individual drugs or target alone | These coating antibodies bind to the drug idiotope, but do not inhibit binding of the drug to its target |
| Paratope-specific, detects free drug | Drug-target complex specific | Detects total drug (free, partially bound, fully bound) |
Krishgen offers a range of ELISA using specific antibodies manufactured using Phage Display technology that allows for accurate estimation of drugs in various stages – bound, free, or total. Learn more about the antibodies we use in our technical note.
ELISA for free and partially bound drug
KBI1018-2 Cetuximab (ERBITUX) ELISA
KBI1026-2 Denosumab (PROLIA) ELISA
ELISA for bound drug complex
KBI1015-4 Adalimumab Antigen Capture ELISA
KBI1017-4 Trastuzumab (HERCEPTIN) ELISA
KBI1019-4 Golimumab (SIMPONI) ELISA
KBI1021-4 Omalizumab (XOLAIR) ELISA
KBI1025-4 Ipilimumab (YERVOY) ELISA
KBI1029-4 Ranibizumab (LUCENTIS) ELISA
ELISA for total drug complex (free, bound, partially bound)
KBI1011-3 Infliximab (REMICADE) ELISA
KBI1013-3 Etanercept (ENBREL) ELISA
KBI1019-3 Golimumab (SIMPONI) ELISA
KBI1022-3 Tocilizumab (ACTEMRA) ELISA
KBI1029-3 Ranibizumab (LUCENTIS) ELISA
