Electrophoretic mobility shift assay (EMSA) as an alternative approach for the estimation of plasma protein binding of oligonucleotides

In this interview, Wenbin Liu (QPS; DE, USA) walks us through the role of plasma protein binding (PPB) studies in drug development, focusing on methodologies such as the electrophoretic mobility shift assay (EMSA) and optimized ultrafiltration method. Liu explores the key considerations of designing a PPB study for oligonucleotides compared to small molecules, offers advice for overcoming challenges in the application of EMSA, and provides his insights on the future of PPB studies.

Could you describe the role of plasma protein binding studies in drug development?

PPB is one of the most fundamental parameters in drug metabolism and pharmacokinetics, needed to understand potential pharmacokinetic/pharmacodynamic (PK/PD) relationships, predict drug-drug interactions and evaluate the toxicity of drug candidates. PPB can affect the disposition (e.g., tissue distribution, clearance, biological half-life, etc.) because the primarily unbound drug concentration is subject to tissue distribution by passive diffusion and elimination through metabolism or excretion. PPB data offers valuable insights into the interactions between drugs and proteins, helping scientists optimize therapeutic designs, improve diagnostic methods and advance our understanding of cellular processes.

How do you interpret plasma protein binding data to assess free fractions, drug exposure and potential toxicity?

The free fraction (fu) represents the unbound portion of a drug in plasma, which is pharmacologically active. A lower fu or a highly protein-bound drug means less free drug is available for interaction with receptors, metabolism and clearance. A higher fu or low protein binding suggests more active drug is available for distribution, metabolism and elimination. High protein binding can limit drug distribution, resulting in low drug exposure and keeping the drug in circulation rather than in tissues. If fu increases, hepatic or renal clearance may also increase, potentially shortening the drug’s half-life and requiring dosage adjustments. A drug with high protein binding can be displaced by another highly protein-bound drug, leading to a potential increase in free drug levels and potential toxicity. Conditions such as liver disease (decreased albumin levels) or kidney disease (altered protein levels) can increase fu, leading to enhanced drug effects and toxicity.

What are some of the key considerations when designing a plasma protein binding study for novel small molecule drugs?

A well-designed PPB study should incorporate robust experimental techniques, appropriate drug concentrations and physiological conditions.

Firstly, selecting the appropriate experimental technique is crucial, as different methods have distinct strengths and limitations. For example, equilibrium dialysis is the most commonly used and reliable method for drugs with moderate-to-low binding but is challenging for drugs that are extremely highly bound, highly lipophilic, insoluble, or have a long equilibrium duration. Ultrafiltration is faster than equilibrium dialysis but may cause non-specific binding to filters. Ultracentrifugation is suitable for highly bound drugs but is challenging for large molecules due to free drug sedimentation and sampling of the free drug layer.

Secondly, the drug concentration should be selected within therapeutic and physiological ranges. Very high concentrations may saturate binding sites and yield misleading fu values.

Additionally, the selection of species (human vs. preclinical species), plasma source (healthy vs. disease-states), plasma pH (acidic vs. basic) and proteins (albumin, alpha-1-acid glycoprotein, lipoprotein) are also important considerations for PPB study design.

What are some of the key differences when designing a plasma protein binding study for small molecules vs. oligonucleotides?

PPB of small molecules relies on classical techniques such as equilibrium dialysis, ultrafiltration and ultracentrifugation, whereas oligonucleotides require specialized methodology due to their size, charge, lipophilicity and binding mechanisms. For oligonucleotides, equilibrium dialysis is not a viable method, as their large molecular size prevents efficient diffusion through the dialysis membrane. The ultrafiltration method can result in an underestimated fu value because the high charge density leads to strong non-specific binding to the filter membranes. The ultracentrifugation method also has limitations, such as free drug sedimentation at extremely high centrifugation speeds. Currently, PPB methodologies for oligonucleotides include the optimized ultrafiltration method and the EMSA. The ultrafiltration method requires a 50 kDa molecular weight cut-off filter to enable adequate recovery and separation of the free fraction from the bound fraction. However, if the oligonucleotides bind to small plasma proteins, the free fraction may be overestimated. This method also encounters recovery issues due to non-specific binding, which must be addressed via pre-treatment of filter surfaces with detergent – though this may impact protein binding. The EMSA is a technique used to detect RNA–protein interactions. Principally, the unbound RNA has specific mobility on non-denaturing gels, whereas RNA bound by protein exhibits reduced mobility. However, the EMSA approach requires dilution steps into phosphate buffered saline and EMSA gel loading solution, which may disrupt equilibrium before measurement.

How do you handle challenges, such as non-specific binding, single stranded vs. double stranded, and major differences in molecule weight, with respect to the application of EMSA to oligonucleotides?

PPB studies of oligonucleotides require specialized methods due to the differences in the physiochemical properties and protein binding properties including the diversity in size, charge, chain length, composition, melting temperature, conjugation and single strand vs. double strand. Addressing challenges in PPB studies, such as non-specific binding, requires appropriate method selection, further optimization and careful data interpretation. Highly charged oligonucleotides can bind non-specifically to filters, dialysis membranes and lab plastics, leading to an overestimation or underestimation of the free fraction. The appropriate blockers, such as Tween or CHAPS, can potentially be applied to reduce non-specific binding. Although current PPB data shows good comparison between ultrafiltration and EMSA at the clinically relevant concentration, more experiments with different generations and classes of oligonucleotides are needed to understand whether EMSA will be a better approach to estimate oligonucleotide fu.

Can you describe how plasma protein binding studies have evolved over your career and where you see the field potentially heading?

I have been working on protein binding for 20+ years and have encountered and overcome numerous challenges in delivering reliable protein binding data. The selection and optimization of methodologies—including equilibrium dialysis, rapid equilibrium dialysis (RED) device, ultrafiltration and ultracentrifugation—along with handling challenging compounds such as those with stability issues, high non-specific binding or extremely high protein binding, are major considerations. With the explosion of oligonucleotide therapeutics over the past 10 years, and the selection of PPB techniques, such as membrane pore size and membrane availability, EMSA application for studying oligonucleotides has presented additional challenges to our daily work life.

The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of Bioanalysis Zone or Taylor & Francis Group.

This content was produced in association with QPS Holdings LLC.