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Plasma protein binding (PPB) of drug candidates can be measured in vitro using plasma from multiple species, including human. The free fraction obtained from in vitro PPB experiments is frequently used in combination with intrinsic clearance to guide structural design and prioritize compounds for downstream in vivo experiments. Comparing free fraction in a range of drug concentrations helps to interpret preclinical PK and PD results and predict the properties of a drug candidate. The measurement of PPB is often performed in conjunction with red blood cell (RBC) partitioning in that both partitioning of free drug into RBC and PPB will affect the free drug concentration available for target binding and, potentially, the pharmacological effect.

Table 1. General comparison of protein binding and drug action.

Highly Protein BindingMinimally Protein Binding
Poorer tissue penetrationBetter tissue penetration
Decreased access to privileged sitesEnhanced access to privileged sites
Slower excretionFaster excretion
Longer plasma half-lifeShorter plasma half-life

Our Detection Methods

Creative Bioarray offers a panel of protein binding services enabling you to better understand the plasma protein binding characteristics of your compounds. With state-of-the-art equipment and scientific expertise in ADME, we are focused on providing cost-effective, high-quality, reproducible data and flexible solutions with fast turnaround times.

  • Rapid equilibrium dialysis method for small molecules
  • Ultrafiltration method for unstable compounds
  • Cross filtration method for larger peptides and biomolecules
  • Ultracentrifugation method
  • Analysis with LC/MS/MS

Equilibrium dialysis is the classic method for assessing plasma protein binding as non-specific binding is minimized compared with other methods such as ultrafiltration, but the process is relatively slow. If a drug or a metabolite is unstable in plasma, this technique cannot be used and ultrafiltration may be a better alternative. Ultrafiltration is suitable for fast screening and requires very little compounds, but this method can be susceptible to non-specific binding. Ultracentrifugation requires a long centrifugation time, which is quite expensive and not commonly used. Another critical aspect of PPB experiments is adsorption. Adsorption at membranes and on the surfaces of devices has a significant impact on the measurement results. Therefore, the mass balance should be evaluated carefully in the PPB experiments.

Creative Bioarray has a choice of four methods combined with three different percentages of plasma (10% plasma, 50% plasma, 100% plasma) to provide flexibility depending on budget and compound characteristics. The application of each option is described in the table below.

Table 2. Applications of the four methods based on different plasma concentrations.

OptionApplicable Situation
10% plasma
  • Reduced plasma requirement and cost.
  • Highly automated evaluation of large numbers of compounds for early screening.
  • Ideal for differentiating between very highly bound compounds.
  • Not suitable for highly unbound compounds.
50% plasma
  • Reduced plasma requirement and cost.
  • Highly automated evaluation of plasma protein binding using a higher concentration of plasma.
  • Recommended for differentiating between highly unbound compounds.
100% plasma
  • "Gold standard" assay.
  • Evaluation of protein binding using 100% plasma.
  • Applicable to all stages of preclinical ADME.

Plasma protein binding can be used to help you prioritize compounds for further development. Our protein binding assays are available for testing in standard human and animal plasma, and can be ordered individually or combined.

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