HSA Biomimetic Chromatography: Rapid Determination of Fraction Unbound (f_u) in PPB Studies for Challenging Compounds

Plasma protein binding (PPB) refers to the degree to which a drug binds to proteins within blood plasma. Fraction unbound (f_u) is a critical DMPK parameter that needs to be measured accurately, because it has significant impacts on the prediction of drug-drug interactions (DDI), estimations of therapeutic indices (TI), and development of PK/PD relationships^[1]. Currently, the primary methods for determining f_u in plasma in the early drug discovery stage include equilibrium dialysis (ED), ultracentrifugation (UC), and ultrafiltration (UF). However, conventional techniques frequently encounter difficulties with certain compounds that are highly lipophilic, exhibit poor plasma stability, or are prone to non‑specific binding (NSB). Mechanistic issues—such as adsorption to the dialysis membrane, long incubation time, and degradation during centrifugation—can lead to inadequate analyte recovery, making accurate f_u determination challenging^[2,3] (as shown in Table 1). Furthermore, for drugs that are highly bound to plasma proteins, analytical methods with extremely high sensitivity are required. This article introduces a novel method for f_u determination—human serum albumin (HSA) biomimetic chromatography—and describes its basic principles, experimental method, calibration model, and applications for compounds that are difficult to assess using traditional methods.

Table 1. Common methods for determining PPB

What is HSA Biomimetic Chromatography

Biomimetic chromatography is the name of the High Performance Liquid Chromatography (HPLC) methods that apply stationary phases containing proteins and phospholipids that can mimic the biological environment where drug molecules distribute. Compared to traditional chromatography, biomimetic chromatography more authentically reflects the interactions between drugs and biological components, providing strong support for predicting pharmacokinetic behaviors in vivo ^[4].

Commonly used biomimetic stationary phases include human serum albumin (HSA), α1-acid glycoprotein (AGP), and immobilized artificial membranes (IAM). All of which have proven valuable in early drug discovery. Over the past two decades, numerous predictive models for volume of distribution, tissue partitioning, protein binding, and blood-brain barrier distribution have been developed using the biomimetic chromatography method. This methodology has been adopted by pharmaceutical companies, including GSK, to investigate hundreds of thousands of compounds^[4,5].

HSA is the most abundant plasma protein, accounting for 55% of the total plasma protein content. It serves as the primary carrier for drug binding to plasma proteins and exhibits high binding affinity for acidic, neutral, and some basic drugs. Given its high abundance in plasma and extensive ligand-binding characteristics, this study focuses on HSA biomimetic chromatography ^[6].

Principles of the HSA Biomimetic Chromatography

HSA biomimetic chromatography employs a stationary phase immobilized with HSA in a high-performance liquid chromatography (HPLC) system to mimic the in vivo protein-binding environment for drugs. When an analyte flows through the column in a mobile phase mimicking physiological conditions (e.g., pH 7.4 buffer), its retention behavior is determined by the binding-dissociation kinetics between the analyte and the immobilized HSA (as shown in Figure 1). Generally, molecules with stronger binding capabilities to HSA have longer retention times on the HSA column, while those with weaker binding have shorter retention times ^[7]. By establishing a correlation model between the drug's retention time in this biomimetic system and its PPB, f_u can be predicted using a simple, rapid chromatographic experiment.

Figure 1. Compound retention depends on interactions between the mobile phase and the stationary phases

Experimental Method of the HSA Biomimetic Chromatography

This method does not require incubation in plasma. The analyte stock solution is diluted to an appropriate concentration using a mixture of ammonium acetate solution (pH 7.4) and isopropanol. After thorough mixing, the sample is analyzed by LC-MS/MS to determine the retention time (t_R) of the compound on the biomimetic HSA column.

Table 2. Equations for calculating f_u in HSA biomimetic chromatography^[8]

Calibration model of the HSA Biomimetic Chromatography

Ten commercially available compounds with human PPB ranging from 5% to 99.5% were selected, covering drugs with low, medium, and high binding. The results demonstrated a strong linear correlation (R² = 0.9923) between the logarithm of retention time and the linearized %PPB (log K) derived from literature-reported human PPB (as shown in Figure 2).

Figure 2. Correlation between retention time and PPB

Key Features of the HSA Biomimetic Chromatography

Compared to equilibrium dialysis, ultracentrifugation, and ultrafiltration, HSA biomimetic chromatography offers the following distinct characteristics for assessing drug PPB:

• No plasma incubation required: Simplifies the workflow.

• Robustness: Not affected by non-specific binding or by stability and solubility issues in the plasma matrix.

• Low sensitivity requirement for the analytical method during sample testing, especially for drugs with high protein binding.

It should be noted that the data reported in the literature reflect total PPB, whereas HSA biomimetic chromatography specifically measures the binding of compounds to HSA. Nevertheless, there is an acceptable correlation between the two methods, indicating that this method can be used for compound PPB screening in the early stages of drug discovery. This finding is also supported by results published by Klara Valkó et al.^[9]

Applications of the HSA Biomimetic Chromatography

Case 1: Small Molecule Drug — Captopril

Captopril is an oral ACE inhibitor used to treat hypertension and chronic heart failure. The thiol group in its molecule is prone to oxidation and can form disulfide bonds with itself or endogenous thiol-containing compounds in plasma (such as cysteine and glutathione), resulting in poor stability. This often leads to low recovery and inaccurate f_u measurements in traditional PPB assays, which typically require optimized reduction systems (such as TCEP) for reliable results. In contrast, the biomimetic chromatography method does not require plasma incubation and effectively avoids such stability issues. Validation confirmed that the f_u value obtained by this method is close to that obtained by ultrafiltration in human plasma (as shown in Table 3).

Table 3. Comparison of f_u determination methods for captopril

Case 2: Peptide — Pasireotide

Pasireotide, an orphan drug used for Cushing's disease and acromegaly, is a 14-amino acid cyclic peptide that is prone to non-specific adsorption, leading to low extraction recovery during sample preparation. Additionally, it exhibits high PPB, making quantification of f_u challenging. When biomimetic chromatography was used for f_u determination, the value was highly consistent with those obtained by ultracentrifugation in human plasma (as shown in Table 4). This approach effectively minimizes errors caused by adsorption loss and insufficient sensitivity, thereby ensuring the accuracy of the result.

Table 4. Comparison of f_u determination methods for pasireotide

Conclusion

HSA biomimetic chromatography method for evaluating PPB has been developed and validated in our laboratory for nearly 100 compounds, including both small-molecule drugs and peptides. This method is effective for challenging cases with poor plasma stability or strong non-specific binding, making it a valuable complementary strategy for f_u determination.

WuXi AppTec DMPK possesses a comprehensive technical system for plasma protein binding (PPB) determination, covering equilibrium dialysis (including HTD and RED), ultracentrifugation, ultrafiltration, flux dialysis, competitive dialysis, biomimetic chromatography, and reporter enzyme methods for dynamic protein binding studies. We can conduct protein binding experiments in various biological matrices such as plasma, tissue homogenates, hepatocytes, and liver microsomes. Furthermore, we support testing for compounds with diverse, complex properties, including unstable compounds, highly bound compounds, covalent inhibitors, peptides, PROTACs, siRNAs, ASOs, etc., providing multi-faceted and highly flexible support for drug development.

Authors: Minxia Li, Chunhong Lu, Mengjie Sun, Xinxin Wen, Jie Wang, Xiaotong Li, Lili Xing

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