Protein Binding Assay Methods for Highly Bound and Unstable Compounds
Poster Authors:
Diana Faiers and Laura Tomlinson
Pharmaron UK Ltd, Hoddesdon
Protein Binding Assay Methods
The decision whether to use one method or another for protein binding assays is dependent on the strength of binding and stability of your compound in plasma. Equilibrium dialysis becomes less precise as fu decreases and compounds begin to degrade during lengthy incubations. Pharmaron has utilized flux dialysis not only in human plasma, but has expanded the technique to alternative matrices and preclinical species. Pharmaron’s poster (recently presented at DMDG/GMP/SPS Joint Meeting 2026) illustrates the performance of flux dialysis compared to rapid equilibrium dialysis (RED), the effect of plasma dilution and how difficult compounds were handled.
Download the poster by filling out the form on the right.
Why does ICH M12 change how protein binding assay methods are chosen?
Regulators have traditionally recommended setting fu, p (the fraction unbound in plasma) equal to 0.01 for drugs that are highly bound to plasma proteins. This conservative default was selected to guard against false negative predictions of drug-drug interactions (DDIs). ICH M12 now permits use of an experimentally measured fu,p for drugs of any degree of protein binding, including those greater than 99 percent protein bound, provided that the accuracy and precision of the method has been demonstrated. If that reliability cannot be demonstrated at less than 1 percent, the 1 percent default value is still used.
Capabilities
ICH M12 defines what is needed to demonstrate that your method is accurate and precise at low levels of protein binding. Validation data should be qualified using positive controls that include compounds with known high binding to plasma proteins of relevance to your method. For newer and emerging protein binding assay methods, M12 recommends qualification of the new method against an orthogonal method already established and accepted for use with highly bound compounds. Method qualification is now part of method selection.
Which protein binding assay method suits which compound?
Where does equilibrium dialysis work well?
Equilibrium dialysis is still the reference technique and covers most of the fu range. Limitations of equilibrium dialysis occur for compounds with extremely tight binding, nonspecific adsorption to the dialysis tube, or chemical degradation before equilibrium because each of these factors will result in an overestimate of the measured value compared to the true unbound fraction.
What does flux dialysis do differently?
Flux dialysis derives the unbound fraction from the initial rate at which a compound crosses the dialysis membrane, so a result is obtained before equilibrium is established. Shortening the incubation limits the time available for degradation and reduces the influence of non-specific binding, which is why the approach suits highly bound compounds and newer modalities such as proteolysis targeting chimeras (PROTACs).
What does plasma dilution change?
The amount of matrix available for each study goes down when you dilute plasma. This is good if you have a limited supply of preclinical plasma (think 3Rs: replacement, reduction and refinement.) It’s also good when dilution stabilizes compounds that are unstable in whole plasma. The poster documents how far measured values can be scaled up to represent undiluted conditions using a defined correction.
Key takeaways
- Flux dialysis was compared against rapid equilibrium dialysis to test how closely the two methods agree.
- The technique was applied across preclinical species, extending validation previously reported only in human plasma.
- Structurally diverse compounds were assessed, spanning small molecules and protein degraders.
- Plasma dilution was evaluated as a route to lower matrix requirements while keeping fu determination valid.
- A case study covers a compound that degraded too quickly to measure in whole plasma.
What is in the poster
The poster gives the full cross-species comparison, the method agreement data with recovery values, the dilution scaling results and the case study showing how dilution rescued an unmeasurable compound. Complete the form to download it.
Frequently asked questions
What main protein binding assay methods are there?
Equilibrium dialysis is the gold standard method. Ultrafiltration and ultracentrifugation are also used in certain circumstances. Flux dialysis has also recently become available. Unlike equilibrium dialysis techniques, the initial rate of transfer across the membrane is read, without waiting for equilibrium.
Why must highly bound compounds be analyzedwith a different approach?
When the free fraction falls below about 1 percent, small absolute errors in recovery or nonspecific binding translate into huge relative errors in fu, so both must be accounted for by the method.
Does M12 require a particular protein binding assay method?
No. Provided evidence is given that the chosen method is accurate and precise, has been qualified with positive controls, and for a novel method qualified against an established orthogonal method.
Can plasma protein binding be determined in diluted plasma?
Yes, if the measured values are corrected for dilution with a validated correction, and it can be shown that the scaling factor holds for the compounds of interest.
Learn more about Pharmaron’s flux dialysis data across alternative matrices and preclinical species.
References
1. ICH. M12 Drug Interaction Studies, Step 4 (21 May 2024), Section 7.2.
2. Kalvass JC, et al. Validation of Flux Dialysis Method. Drug Metab Dispos. 2018;46(4):458-469. PMID 29437872.
3. Riccardi K, et al. Plasma Protein Binding of Challenging Compounds. J Pharm Sci. 2015;104(8):2627-2636. PMID 26037784.
4. Jitta SR, et al. Determination of free fraction (fu) by flux dialysis using the RED device. Xenobiotica. PMID 42047399.
5. EMA. ICH M12 drug interaction studies scientific guideline.