Pharmacokinetic (PK) assays are a crucial stage of drug development. They help ensure the safety and effectiveness of new therapeutics by establishing how drugs are absorbed, distributed, metabolized, and excreted in the body.
Nonspecific binding (NSB) can complicate PK assays, often reducing their accuracy and reliability. NSB occurs when the analyte in a solution binds to a solid surface, which can derail PK assays at various stages. The issue can affect the dosage and concentration of a formula and skew results. Fortunately, developers can avoid potentially expensive delays brought on by NSB in PK assays by adopting some of the mitigation strategies below.
What factors contribute to NSB in drug development?
Three significant factors cause NSB during drug development, each of which contributes to the problem in unique ways. All of these factors must be properly addressed to avoid delays to development timelines.
- Material composition of solid surfaces: The differences in material composition of solid surfaces mean they contain various functional groups, leading to distinct interactions with analytes. For example, glassware surfaces are abundant in silanol groups that readily acquire a negative charge. This makes them more likely to bind with positively charged molecules through ionic interactions.
Conversely, polypropylene and polystyrene materials offer different challenges. They are rich in hydrophobic groups, making them prone to binding to hydrophobic molecules. Metal surfaces contain metal cations, making them likely to bind with anionic molecules through ionic interactions.
- Solution composition: Solution components affect the dissociation state and the solubility of compounds. They can also physically adsorb onto solid surfaces, causing issues with PK assays. For example, pH influences the dissociation state of a compound, causing analytes to be present in either dissociated or molecular forms. This affects their interaction with solid surfaces.
Buffer salts in the solution can attach to binding sites on solid surfaces, while protein and lipids in the solution can bind with analytes, weakening or eliminating the analyte binding to solid surfaces.
- Compound properties: Compounds can be one of three types: hydrophobic, hydrophilic, or amphiphilic. Hydrophobic compounds such as alkanes and aromatic bases are rich in hydrophobic groups and primarily bind to solid surfaces with a similarly high proportion of hydrophobic groups.
Hydrophilic compounds contain easily dissociable groups that are either negatively or positively charged and primarily bind to solid surfaces through ionic interactions and hydrogen binding. Amphiphilic compounds possess both characteristics.
Addressing NSB challenges
NSB challenges can be complex, requiring a disciplined approach to address them. First, it’s important to confirm the type of solid surface on which the adsorption occurs. By understanding the compounds and the main forces involved in NSB, you can replace the material or modify the solution composition to weaken or eliminate the physical adsorption of analytes to the solid surface.
If evidence exists of adsorption onto container walls, using low-adsorption consumables or adding desorption agents, such as proteins or surfactants, will typically solve the problem. If adsorption onto the chromatography system occurs, adjust the ionic strength of the mobile phase and column temperature, use inert tubing, or use a column with weaker hydrophobic interactions.
During urine sample collection and processing, analytes that are hydrophobic compounds tend to undergo NSB to the inner walls of plastic containers. This can result in lower measurement values, but using a low-binding material or adding surfactant eliminates these influences.
Future challenges
For new molecular entities, such as peptides and oligonucleotides, similar methods are used to avoid container wall adsorption. However, during separation in chromatography, these molecules are prone to adsorb on the solid phase of chromatographic columns, metal filers at the ends of columns, and pipelines. This leads to chromatographic peak tailing, more significant carryover, and lower measured values for low-concentration samples. These issues can be addressed by changing the ionic strength of the mobile phase, using inert tubing, adjusting the temperature of the column, or replacing the column.
Most current research focuses on container walls, but analytes can also bind to cell membrane surfaces, tissue proteins, and other components in different tissue homogenate matrices. More attention must be paid to this type of binding, as it can result in differences in extraction recovery in sample processing, mostly in lower recovery at low concentrations. This can cause a phenomenon similar to instability.
A final word
NSB can compromise the accuracy of PK assays, creating delays and risks in preclinical drug development. Addressing NSB through careful material selection, optimized solutions, and expert strategies is essential to ensure reliable data and smooth progress. Drug developers and sponsors who do not have the capability or capacity to manage NSB and other PK assay challenges would do well to partner with an experienced lab partner who does.
As a global company with operations across Asia, Europe, and North America, WuXi AppTec provides a broad portfolio of R&D and manufacturing services that enable the global pharmaceutical and life sciences industry to advance discoveries and deliver groundbreaking treatments to patients. Through its unique business models, WuXi AppTec’s integrated, end-to-end services include chemistry drug CRDMO (Contract Research, Development and Manufacturing Organization), biology discovery, preclinical testing and clinical research services, advanced therapies CTDMO (Contract Testing, Development and Manufacturing Organization), helping customers improve the productivity of advancing healthcare products through cost-effective and efficient solutions. WuXi AppTec received an AA ESG rating from MSCI for the fourth consecutive year in 2024 and its open-access platform is enabling more than 6,000 customers from over 30 countries to improve the health of those in need – and to realize the vision that “every drug can be made, and every disease can be treated.”
