TRANSIL High Sensitivity Binding Kit
When Conventional Plasma Protein Binding Assays Fail Measure the unbound fraction of compounds with fu < 1%, strong non-specific binding, or poor solubility — even when equilibrium dialysis or ultracentrifugation fail. Key benefits Reliable determination of very low free fractions (fu ≈ 0.001 up to 5%) Works with lipophilic compounds prone to non-specific binding or poor solubility 30-minute incubation enables measurement of unstable compounds High compound recovery despite nonspecific binding Compatible with plasma from any species When to Use the TRANSIL High Sensitivity Binding Kit This assay is particularly useful when conventional plasma protein binding methods produce unreliable results. Use the HSB assay if your compound: shows very strong plasma protein binding (fu < 1%) exhibits high lipophilicity or amphiphilicity shows low recovery in dialysis or ultrafiltration exhibits strong non-specific binding to membranes or plastics is unstable during long dialysis incubations Typical Compounds That Benefit From the HSB The TRANSIL High Sensitivity Binding assay was designed for compounds whose physicochemical properties make plasma protein binding difficult to measure using conventional techniques. Highly lipophilic small molecules Compounds with high logP/logD that strongly partition into lipid membranes. Amphiphilic and membrane-interacting molecules Compounds containing both hydrophobic and polar regions that interact strongly with phospholipid bilayers. Beyond Rule-of-5 (bRo5) compounds and degraders Large, flexible molecules such as PROTACs and other heterobifunctional degraders that often exhibit high lipophilicity and problematic behavior in conventional PPB assays. Macrocycles and peptide-based modalities Macrocycles, cyclic peptides, lipidated peptides, and peptide fragments with hydrophobic surfaces or membrane-interacting motifs. Highly hydrophobic tool compounds or screening hits Compounds that show strong nonspecific binding to plastics, membranes, or other assay components. These modalities often exhibit very strong apparent binding or low recovery in traditional techniques such as equilibrium dialysis or ultrafiltration. The membrane phase used in the HSB assay provides a robust equilibrium shift that allows reliable determination of the unbound fraction even for such difficult compounds. Limitations of Conventional Plasma Protein Binding Assays Many modern drug modalities — particularly highly lipophilic, amphiphilic, or beyond- Rule-of-5 (bRo5) compounds such as PROTACs and macrocycles— often exhibit extensive non-specific binding in conventional plasma protein binding assays. In equilibrium dialysis or ultrafiltration systems, these molecules often interact not only with plasma proteins but also with assay components such as membranes, plastic labware, and other hydrophobic surfaces. As a result, a significant fraction of the compound may adsorb to these materials rather than remaining in solution, leading to low recovery, distorted equilibria, and inaccurate estimates of the unbound fraction. Very strongly bound compounds present an additional challenge because the free fraction becomes extremely small. When the unbound fraction (fu) approaches 0.1% or lower, only minute amounts of compound are present in the free phase measured in equilibrium dialysis or ultracentrifugation. Under these conditions, the analytical signal approaches the lower limit of quantification of the LC-MS method, so even small experimental errors or compound losses can result in large relative errors in the calculated fu value. Equilibration can also be slow for large or complex molecules such as PROTACs, macrocycles, or peptide-based modalities. These compounds diffuse more slowly and cross dialysis membranes less efficiently than small molecules, often requiring long incubation times to reach equilibrium. Incomplete equilibration can therefore lead to systematic errors in the measured free fraction. Modern drug discovery increasingly produces lipophilic and beyond-Rule-of-5 molecules that are difficult to analyze with conventional plasma protein binding assays. The TRANSIL High Sensitivity Binding Kit was designed specifically for these challenging modalities. TRANSIL High Sensitivity Binding Approach The TRANSIL High Sensitivity Binding (HSB) assay is a competitive equilibrium shift assay that determines plasma protein binding by measuring the distribution of a compound between phosphatidylcholine membranes immobilized on TRANSIL beads and plasma proteins in solution. The assay concept is derived from the classical erythrocyte partitioning method, where plasma proteins compete with cell membranes for binding of the drug candidate. In the HSB assay, synthetic phosphatidylcholine membranes immobilized on beads replace erythrocytes, providing a standardized and easy-to-handle membrane phase while maintaining the same underlying binding principle. Importantly, plasma is present in every assay compartment, stabilizing compounds in solution and minimizing artefacts caused by precipitation or surface adsorption. The assay is therefore conceptually similar to a highly controlled erythrocyte partitioning assay, but implemented in a standardized micro-assay format that eliminates the need to isolate and handle unstable erythrocytes. Binding affinity is determined by systematically shifting the binding equilibrium between plasma proteins and the membrane phase. This is achieved by combining five different membrane surface areas with five plasma dilutions, generating a matrix of equilibrium conditions. The resulting changes in the apparent membrane affinity are analyzed using a mechanistic binding model to determine the compound’s affinity to plasma and the corresponding free fraction. A key feature of the method is that no direct measurement of the unbound drug concentration is required. Instead, the assay only quantifies the compound remaining in the liquid phase after removal of the membrane beads, which contains both free compound and compound bound to plasma proteins. The free fraction is then calculated from the way the binding equilibrium shifts across the plasma dilution series. Like other TRANSIL assays, the HSB assay incorporates multiple internal quality control measures, including reference wells, replicate determinations across membrane surface areas, recovery monitoring, and model-based consistency checks. These controls ensure robust results even for compounds with very strong binding, poor solubility, or high non-specific adsorption. How the Assay Works The assay determines plasma protein binding affinity through the following experimental workflow: Run membrane affinity pre-test to determine optimal plasma dilution steps Prepare plasma dilution series Add plasma and compound Incubate 30 minutes (or 120 mixing cycles) Remove membrane beads by centrifugation Quantify compound concentration Calculate plasma binding Measures extremely strong plasma protein binding Equilibrium shift assay design enables accurate determination of very low free fractions (fu well below 1%). This enables reliable plasma protein protein binding measurements for compounds where conventional assays often fail due to analytical limitations. Works with highly lipophilic and “sticky” compounds Phosphatidylcholine membranes immobilized on TRANSIL beads provide a physiologically relevant lipid phase that acts as a controlled binding sink. Accurate measurements for difficult modalities such as lipophilic small molecules, PROTACs, peptide fragments, and amphiphilic drugs. Robust against non-specific binding artefacts All assay compartments contain plasma and silanized glass vials (or ultra-low binding wells) minimize surface adsorption. Improved recovery and reproducibility even for compounds that show strong non-specific binding to plastics or membranes. No direct measurement of the free fraction required Binding affinity is derived from the equilibrium shift across a matrix of membrane surface areas and plasma dilutions. This avoids the analytical challenges associated with detecting extremely low free drug concentrations. Short incubation time Equilibrium is typically reached within 30 minutes. Enables analysis of compounds that may be unstable during long dialysis incubations. Mechanistic binding model Data are analyzed using a physico-chemical binding model describing competition between plasma proteins and membranes. Produces robust free fraction estimates and binding parameters across a wide range of compound properties. Built-in internal quality controls The assay includes reference wells, recovery checks, replicate measurements, and model-based consistency metrics (TRANSIL Quality Index). Ensures high confidence in results and allows rapid identification of problematic measurements. Automation-compatible workflow Simple liquid handling steps and centrifugation-based phase separation. Can be easily integrated into automated ADME screening workflows. Broad applicability across drug modalities Assay design accommodates compounds with very strong plasma binding, poor solubility, or strong membrane affinity. Suitable for modern drug discovery programs including bRo5 compounds, degraders, peptides, and complex small molecules. Use the TRANSIL Equilibrium Shift Assay for compounds with low membrane affinity. Validation Against Literature and Erythrocyte Binding Assay The performance of the TRANSIL High Sensitivity Binding (HSB) assay has been evaluated using both reference compounds and challenging discovery molecules, and the results were compared with established plasma protein binding methods. For drugs with typical physicochemical properties, the free fractions determined with the HSB assay closely match values obtained with equilibrium dialysis, demonstrating that the assay provides results consistent with widely used reference techniques. For compounds exhibiting very strong plasma protein binding, results obtained with the HSB assay are also in excellent agreement with measurements from the erythrocyte partitioning method, which has historically been used to overcome limitations of dialysis-based techniques. Importantly, the HSB assay can reach comparable equilibrium results within 30 minutes, whereas dialysis experiments with highly lipophilic compounds may require extended incubation times to converge to similar free fraction estimates. In addition to accuracy, the assay shows excellent reproducibility, as illustrated by repeated independent measurements of diclofenac. Together, these results demonstrate that the TRANSIL HSB assay provides accurate, robust, and reproducible determination of plasma protein binding, including for compounds that are difficult to analyze using conventional methods. Figure 1: Comparison of fraction unbound measurements obtained via dialysis and the TRANSIL High Sensitivity Binding kit. Marketed drugs having average physchem properties yield comparable results in both assays Figure 2: Comparison of fraction unbound measurements obtained via erythrocyte partitioning and the TRANSIL High Sensitivity Binding kit. Early discovery compounds binding strongly to plasma proteins yield comparable results in both assays. Figure 3: Comparison of fraction unbound measurements at 4, 6, and 24 hours incubation in dialysis versus 30 minutes incubation in the TRANSIL High Sensitivity Binding kit (dotted line). The compound is a lipophilic acid with a cLogP of 6.1 and a molecular weight of 503. Fraction unbound estimates from the longer incubation times of dialysis converge towards the result of the TRANSIL assay. Figure 4: Reproducibility of the TRANSIL High Sensitivity Binding kit showing results from 5 independent fraction unbound measurements of diclofenac. How is the TRANSIL High Sensitivity Binding Kit different from the standard 96-well TRANSIL kits? The Transil High Sensitivity Binding (HSB) kit is different from all our other screening kits, in that it is designed to measure plasma binding of compounds that are otherwise very difficult – if not impossible – to handle in dialysis or ultracentrifugation. This kit measures plasma binding of only one compound. It does that by establishing 25 different equilibrium binding states. They are characterized by 5 different plasma dilutions crossed by 5 different immobilized membrane surface areas that create a competitive binding substrate for your test items. There is an exact formula for describing this competitive binding situation and the supplied spreadsheet uses it to numerically derive the plasma binding parameters. This approach allows you to have plasma in every assay compartment, so that the solubility is increased, and non-specific binding decreased. The assay kit supplies everything needed for the measurement, except plasma. How does the TRANSIL High Sensitivity Binding Kit work? The Transil High Sensitivity Binding (HSB) kit measures plasma binding of only one compound. It does that by establishing 25 different equilibrium binding states. They are characterized by 5 different plasma dilutions crossed by 5 different immobilized membrane surface areas that create a competitive binding substrate for your test items. There is an exact formula for describing this competitive binding situation and the supplied spreadsheet uses it to numerically derive the plasma binding parameters. This approach allows you to have plasma in every assay compartment, so that the solubility is increased, and non-specific binding decreased. How is the TRANSIL High Sensitivity Binding Kit different from the TRANSIL PPB kit? Those two kits are very different. The TRANSIL PPB Binding Kit is a fast screening kit for plasma protein binding. It contains both HSA and AGP coated beads, mixed at a physiological ratio. While that kit comes in very high quality polypropylene (made my micronic) with very low non-specific binding, there is still a measurable difference to the non-specific binding in the silanized glass vials of the TRANSIL High Sensitivity Binding (HSB) Kit. The beads used in the HSB kit are coated with phosphatidylcholine membranes and the assay is utilizes our equilibrium shift assay principle based on competitive binding to plasma (in solution) and membranes (immobilized). This kit is our specialty solution for all compounds that either (i) bind too strongly to plasma that it’s difficult to quantify, (ii) have low solubility in pure buffer and thus precipitate in normal test kits that have assay compartments without plasma, (iii) exhibit high non-specific binding, or any combination of that. The price per compound of the TRANSIL High Sensitivity Binding Kit is substantially higher than alternative means, however, they get you results when the compounds are difficult. Why is the solubility and recover in the TRANSIL High Sensitivity Binding Kit higher than in any other assay system? All assay compartments of the Transil High Sensitivity Binding (HSB) kit contain plasma which increases compounds’ solubility and decreases non-specific binding. Also, the HSB kit comes is silanized glass vials that further suppress non-specific binding. As some compounds (e.g. peptides) bind strongly to silanized glass, this kit is also available in low-binding polypropylene wells. Can the TRANSIL High Sensitivity Binding Kit be used for peptides? Yes, we offer a variant of the kit in 96-well format with low-binding polypropylene tubes. How critical is plasma instability in the TRANSIL High Sensitivity Binding Kit? The compounds are incubated at room temperature. This reduces esterase activity. And since the incubation time is only 30 minutes, there is only minimal compound degradation due to esterases. In fact, the incubation time can be reduced to 10 minutes when compounds have normal on and off rates.