Overcoming nonspecific binding challenges in pharmacokinetic assays

However, nonspecific binding (NSB) can hinder PK assays, impacting their accuracy and reliability. Fortunately, there are strategies and best practices to counter this issue. 

NSB occurs when analytes in a solution bind to a solid surface. This can happen at various stages of PK assays and can potentially skew results by affecting a formula’s dosage and concentration. Solid surfaces, solution composition, and compound properties are the primary contributors to NSB. Achieving accurate results requires extensive knowledge of these factors and experience working with them.

Peiyun An, an associate director in WuXi AppTec’s DMPK department, explains the mechanics of NSB, how it affects PK assays, and the best methods and techniques for addressing the issue. 

Can you explain how NSB can impact PK assays? ​

Physisorption can cause a solution’s analytes to bind to solid surfaces, resulting in NSB. Binding can happen due to hydrophobic forces, ionic interactions, van der Waals forces, and hydrogen bonding. NSB reduces the concentration in the solution. NSB issues can emerge at various stages of the PK experimental process, including formulation preparation, administration to experimental animals, sample collection and storage, and during the analyzing of biological samples. NSB affects the concentration of the formulation and the dosage administered but also impacts the true concentration of biological samples, compromising the accuracy and reliability of PK assay results.  

What are the main factors contributing to NSB in drug development?​

Each factor i.e., solid surfaces, solution composition, and compound properties—contributes to NSB in different ways. For example, solid surfaces contain various functional groups, which lead to distinct interactions with analytes due to differences in material composition.

Glassware surfaces are abundant in silanol groups, which readily acquire a negative charge, making them likely to bind with positively charged molecules through ionic interactions. In contrast, polypropylene and polystyrene materials are rich in hydrophobic groups, which makes them prone to binding with hydrophobic molecules via hydrophobic forces. Metal material surfaces contain metal cations, which make them inclined to bind with anionic molecules through ionic interactions. 

Solution components affect the compounds’ dissociation state and solubility and can also physically adsorb onto solid surfaces. For example, pH influences the dissociation state of compounds, causing analytes to exist in either dissociated or molecular forms, which affects their interaction with solid surfaces. Buffer salts in the solution can compete for binding sites on solid surfaces, while proteins and lipids in the solution may interact with analytes, weakening or preventing their attachment to these surfaces. 

Compounds can be hydrophobic, hydrophilic, or amphiphilic. Hydrophobic compounds rich in hydrophobic groups, such as alkanes and aromatic bases, primarily bind to solid surfaces that are also rich in hydrophobic groups. Hydrophilic compounds contain easily dissociable groups that carry a negative or positive charge—e.g., ammonium, carboxyl, phosphate, and sulfonate groups—and primarily bind to solid surfaces through ionic interactions and hydrogen bonding. Amphiphilic compounds possess both characteristics.   

How does WuXi AppTec currently approach NSB issues, and what best practices or methodologies have proven most effective? ​

NSB issues can be quite complex. First, it’s necessary to confirm which type of solid surface the adsorption occurs on. Then, by understanding the properties of the compounds and the main forces involved in NSB with solid surfaces, we can replace the material or modify the solution composition to eliminate or weaken the physical adsorption of the analytes to the solid surface.

When there’s evidence of adsorption onto container walls, the problem can typically be solved using low-adsorption consumables or adding desorption agents—e.g., surfactants or proteins. For adsorption onto the chromatography system, we can adjust the ionic strength of the mobile phase, change the column temperature, use inert tubing, or replace the column with one with weaker hydrophobic interactions. 

Are there any aspects of NSB that are inadequately addressed in the existing literature? ​

Current literature mainly focuses on the container walls in contact with analytes. However, this ignores the fact that analytes can bind to cell membrane surfaces, tissue proteins, and other components in different tissue homogenate matrices. This binding can result in differences in extraction recovery at low and high concentrations in sample processing, with notably lower recovery at low concentrations. This can cause a phenomenon similar to instability, making it essential to establish during the tissue homogenous method development process.  

Can you share examples of NSB challenges you’ve encountered, and how they were resolved? Would your answer change if we were talking about new modality drugs?  ​

When the analytes are hydrophobic compounds, they tend to undergo NSB to the inner walls of plastic containers during urine sample collection and processing, which can result in lower measurement values. We usually use low-binding material or add an appropriate amount of surfactant to eliminate such influences. 

For some new molecular entities, such as peptides and oligonucleotides, these methods also address container wall adsorption. However, during the separation process in chromatography, these entities are prone to adsorb on the solid phase of the chromatographic column, the metal filters at both ends of the column and pipelines leading to problems such as chromatographic peak tailing, larger carryover, and lower measured values for low-concentration samples. 

We can address these issues by changing the ionic strength of the mobile phase, using inert tubing, adjusting column temperature, or replacing the column with one that has weaker hydrophobic interactions.

A final word​

NSB issues can seriously affect the accuracy and reliability of PK assays, potentially costing drug developers time and money as they seek to bring new drugs to the market and into patients’ hands. By properly understanding the NSB process, developers can avoid muddled results that affect development timelines. Partnering with an experienced and knowledgeable lab partner is the easiest way to ensure NSB doesn’t negatively impact a drug’s journey to market.