Nonspecific Binding: Main Factors of Occurrence and Strategies

Nonspecific binding is a form of adsorption resulting from non-covalent bonding forces. In a solution, the test article will bind to the solid surface due to the electrostatic interactions or the hydrophobic effect of the compound. The nonspecific binding could occur in the whole process of pharmacokinetic (PK) study, such as formulation preparation, dosing, sample collection, storage, and analytical testing of biological samples. Adsorption not only affects the results of the formulation and sample analysis but also leads to inconsistent sample extraction recovery calculations of standard curves at different concentrations, resulting in higher signal intensity at high-concentration and lower signal intensity at low-concentration. During instrumental analysis, adsorption can also affect the chromatographic peak shapes and leads to system carryover, thereby affecting the accuracy of the results. This article discusses the common approaches and ideas for solving adsorption problems based on the principles of nonspecific binding and our experiences.

Three Factors of Nonspecific Binding

The quantity and strength of adsorption of test articles are closely related to three factors: the solid surface in contact with the solution, the composition of the solution, and the properties of the compound. These three elements will be described below in relation to practical scenarios of pharmacokinetic assays, including drug dosing, sample collection, and sample analysis (see Figure 1).

Figure 1. Three factors of nonspecific binding

Types of solid surfaces

During formulation preparation, sample collection, and sample analysis, compounds will be exposed to the solid surfaces of various materials. The main types of solids include glassware, plastic consumables, as well as phase lines and columns of liquid chromatography. Each material exhibits different adsorption principles, as summarized in Table 1.

Table 1. Adsorption principles of different material surfaces¹

Type of solution

Solvents used for dissolving standard analytes or preparing formulations are composed of water, organic solvents, or dispersants. Small molecule analytes are highly soluble in these solvents, and the adsorption are relatively weak. However, biological matrices consist of complex components. For instance, in addition to a large amount of water, plasma also contains plasma proteins and lipids such as phospholipids. Small molecule drugs may bind to plasma proteins, and lipids could also attenuate the adsorption of the analyte. Consequently, in matrices such as whole blood, serum, and plasma, the adsorption of analytes is relatively weak. ² In contrast, fluids such as urine, bile, and cerebrospinal fluid have lower concentrations of proteins and lipids, which increases the possibility of nonspecific adsorption during sample collection and bioanalysis.

Type of analytes

Some small molecule drugs may exhibit adsorption in excretory samples such as urine and bile, as well as in matrices such as cerebrospinal fluid. Notably, large molecule drugs such as peptides, proteins, peptide-drug conjugates (PDC), nucleic acid drugs, and cationic lipids are more likely to exhibit adsorption, which is influenced by the structure of the drug molecule. Peptides, proteins, and PDCs primarily consist of amino acids that are amphoteric compounds with both positively charged amino groups and negatively charged carboxyl groups, resulting in strong electrostatic effects. ³

The R group of some amino acids such as lysine, arginine, and histidine, separately have additional positively charged groups: amino groups, imidazolyl group, and guanidinyl groups (see Figure 2). Besides, owing to the relatively large structure, these molecules such as peptides, proteins, and PDCs exhibit pronounced electrostatic and hydrophobic effects. Therefore, these molecules may also be affected by adsorption in the matrices such as urine, bile, and plasma, as well as in the process of preparation of working solutions containing only reagents.

Figure 2. Adsorption of some amino acid residues with positively charged groups generating electrostatic interaction

Nucleic acids are amphoteric molecules composed of bases, ribose or deoxyribose, and phosphoric acid. Pyrimidines and purines contain large amounts of amino groups, and phosphate groups easily bind to metal surfaces (see Figure 3).⁴

Figure 3. Schematic diagram of five common bases and structures of nucleic acids

Cationic lipids also possess amphiphilic properties, as exemplified by 2,3-dioleoxypropyl trimethylammonium chloride (DOTAP). DOTAP consists of a head group containing a positively charged quaternary ammonium salt, connected to a long chain tail through an ester bond linking fragment. The head group induces a strong electrostatic effect, while the tail generates a hydrophobic effect, leading to pronounced adsorption (see Figure 4).⁵

Figure 4. Structure of cationic lipid DOTAP. The head group and tail have electrostatic and hydrophobic effects respectively

Other factors affecting adsorption

The three factors of nonspecific binding determine the occurrence of adsorption. In addition to these three factors, some other factors such as ambient temperature, solution pH, exposure time of the solution to the solid surface, and even the number of freeze–thaws the solution undergoes will impact the degree of adsorption. Consequently, these factors need to be examined during the actual assay process.

Common Approaches to Solve Non-specific Binding

The larger the contact surface area and the longer the contact time between the analytes and the surfaces of consumables or pipelines in the solution, the more severe adsorption becomes.⁶ This principle underlies common methods for investigating nonspecific adsorption, including continuous transfer and gradient dilution. Additionally, the presence and extent of adsorption can be determined by comparing the signal differences when the same volume of solution is placed in containers of different sizes or when different volumes of solution are placed in containers of the same size. For different types of biological matrices, desorption agents are typically added to facilitate the process of desorption. General desorption approaches for various biological matrices are summarized in Table 2.

Table 2. General desorption pathways for different biological matrices

The surfactant has a hydrophilic group at one end and a hydrophobic group at the other end. The addition of surfactants can lead to a more uniform dispersion of analytes in the solution, thereby improving the dissolution state and weakening the hydrophobic effects that produce nonspecific adsorption. ⁷ The addition of surfactants is closely related to varying degrees of signal suppression or interference during the mass spectrometry of analytes. Therefore, suitable surfactants need to be selected for different compounds to provide a desorption effect while avoiding interference with the detection of analytes. Commonly used surfactants are listed in Table 3.

Table 3. Classification of surfactants

During sample collection and pretreatment, the adsorption effect can also be mitigated through the use of low-adsorption consumables, such as low-adsorption tubes designed for proteins and nucleic acid compounds and low-adsorption 96-well plates. During analysis and detection, the use of surface-passivated chromatographic columns and liquid phase systems with low adsorption properties can reduce the adsorption of phosphorylated compounds (see Figure 5) and nucleic acid drugs (see Figure 6). This effect improves the peak shapes and higher mass spectrometry signals.

Figure. 5 A) Structural formula of AMP and ATP. B) Symmetrical peak shapes achieved utilizing low adsorption liquid phase system and chromatographic column. C) Conventional liquid phase system shows significant tailing of peaks.

Figure. 6 A) Structural formula of phosphorothioate-modified nucleic acid analogs. B) a) Conventional and b) low adsorption liquid phase system with nearly 10-fold signal increase signal

Strategies for Addressing Nonspecific Binding in New Modality Drugs

Table 4. Strategies and underlying mechanisms for addressing nonspecific binding in new modality drug types

Peptides, proteins, and PDC drugs exhibit significant adsorption because of their poor solubility. Adjusting the pH of the dissolution solvent and optimizing the type and composition of the dissolution solvent could improve the solubility of the compound to some extent, thereby reducing the adsorption effect. Additionally, these compounds often exhibit low signal response in the liquid-mass system, therefore, careful selection and optimization of desorption agents is crucial to meeting the sensitivity requirements.

We have also built a platform for the detection of nucleic acid drugs, which are typically modified with a phosphorothioate backbone, as shown in Figure 7. This modification enhances the enzyme stability in the matrix and the binding affinity of nucleic acid drugs to plasma proteins, thereby reducing nonspecific adsorption. In addition to using desorption agents, adding chelating agents such as ethylenediaminetetraacetic acid (EDTA) to the mobile phase and adjusting pH can also attenuate the adsorption of nucleic acid drugs on the metallic path and column to some extent. ⁸Finally, a low adsorption column and liquid phase system with passivated metal path surface can be used to reduce the adsorption effect, thereby improving the recovery of nucleic acid drugs and reducing the lower limit of detection.

Figure 7. Phosphorus-sulfur backbone and glycosyl modifications of nucleic acid-based drugs

Conclusion

Nonspecific binding of compounds affects the accuracy of pharmacokinetic assay results. Therefore, selecting appropriate solvents and vehicles based on the characteristics of the compounds under investigation is imperative. Furthermore, optimizing sample collection and storage conditions, developing a suitable pretreatment process for biological samples, and screening the liquid phase system is essential to ensuring the stability of analytes using pharmacokinetic assays. Finally, implementing these measures can ensure the accuracy and reliability of assay results.

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Authors: Minhui He, Zhengzhen Gao, Jinlian Lu, Lingling Zhang, Lili Xing

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