Rapid Equilibrium Dialysis (RED) Device.
A Transforming Technology for Plasma Protein Binding Assays.

Determining the extent to which a molecule binds to plasma proteins is a critical phase of pharmaceutical development because it influences compound dosing, drug efficacy, clearance rate and potential for drug interactions. This determination is enabled by equilibrium dialysis, an accepted standard method for reliable estimation of the nonbound drug fraction in plasma. Although it is the preferred method, equilibrium dialysis is generally labor-intensive, time-consuming, cost-prohibitive and difficult to automate. The RED Device for rapid equilibrium dialysis was developed in close association with the pharmaceutical industry to specifically address these issues, accelerating lead optimization and reducing the attrition rate. In addition to plasma protein binding, the device is used for determining drug partition between red blood cell and plasma, protein binding of liver microsomes to improve the correlation between in vitro and in vivo intrinsic clearance, the competition between tissue protein binding against plasma proteins and dissociation constant determination (Kd). The RED device is available in a molecular weight cutoff (MWCO) of 8,000 Da.

Reusable and Single-Use Plate Rapid Equilibrium Dialysis Systems

The RED Device System utilizes disposable tube inserts. Each insert is made of two side-by-side chambers separated by a vertical cylinder of dialysis membrane (MWCO ~8,000) validated for minimal nonspecific binding. This format requires no extensive assembly steps or specialized equipment, and each chamber/well is easily accessible from the top of the insert after insertion in the base plate. Additionally, the high membrane surface-to-volume ratio allows rapid dialysis, where equilibrium can be reached in 4 hours with high levels of reproducibility and accuracy. Incubation times as low as 100 minutes can be acheived with proper agitation.

RED Device Inserts are designed to be used with either the reusable PTFE or disposable high-density polypropylene base plates. Each RED Device Base Plate holds up to 48 RED Device Inserts and has a standard 96-well plate footprint with 9 × 9 mm well spacing to provide compatibility with automated liquid handling systems. In addition, the disposable RED Device Base Plates are available pre-loaded, providing operation convenience for scientists conducting protein-binding applications. No pre-conditioning of the membrane inserts is needed. When using radioactive materials, this single-use plate is easily disposed of to avoid contamination and cleaning. RED Device Inserts and Base Plates are also available separately.

The RED Device has been extensively validated for plasma-binding assays producing results consistent with those reported in the literature. Using the RED Device to measure Warfarin binding to plasma (human and rat) proteins at two concentrations of 1 and 10 µM, the RED Device produced results with minimal intra-experimental variability.

Rapid Equilibrium Dialysis Device Highlights:

• Ease of use – disposable tubes require no presoaking step, assembly or specialized equipment
• Short incubation time – due to high membrane surface-to-volume ratio, equilibrium can be reached in as little as 100 minutes with proper agitation or in 3-4 hours without agitation
• 96-well format – suitable for automated liquid handlers
• Flexible – can be used for any number of assays (1-48 assays/plate) without wasting the whole plate
• Robust – compartmentalized design eliminates potential for cross talk or leakage
• Reproducible and accurate – validated for plasma-binding assays producing results consistent with those reported in literature
• Versatile – the reusable RED Device Base Plate is composed of chemically inert high grade PTFE, eliminating nonspecific binding and risk of contamination
• Automation friendly – the single-use RED Device Base Plates are lightweight and suitable for automated plate and liquid handling workstations. The RED Device Inserts are designed with large openings to allow better access with a wide range of pipette tips
• Preloaded and disposable plates – single-use RED Device Base Plates do not require a lengthy plate washing step and are available preloaded with inserts that do not require presoaking
• Validated – each lot functionally tested in a protein-binding assay for guaranteed performance

Applications for RED Device:

• Determination of free vs. drug bound to plasma proteins
• Pharmacokinetics studies
• Formulation of drug dosage for in vivo studies
• Drug-drug interaction studies
• Selection criteria during drug lead optimization
• Drug partition between plasma and whole blood
• Solubility study
• Dissociation constant determination (K_d)
• Tissue-binding study using tissue homogenate

Use the RED Device for increased productivity. Each RED Device Base Plate sits in a 96-well plate footprint withe 9 x 9 mm well spacing making it compatible with automated liquid handling systems. Single-Use RED Base Plates are available with inserts preloaded mimimizing setup time. Rapid Equilibrium Dialysis Device Frequently Asked Questions. Please click here to see the Rapid Equilibrium Device Demonstration Video.

Measurement of Plasma Protein Binding: A Comparison of Conventional, 96-well and Rapid Equilibrium Dialysis (RED) methodologies (Opens new broswer window)
Nasreen Malik, Anthony Glazier, Fiona Henderson, Sarah Andrews, Georgina Adjin-Tettey, Anthony Chadwick and David Lankaster.
Department of DMPK, Covance Laboratories, Harrogate, UK

Characterization of Drug Distribution in Tissues Using a Rapid Equilibrium Dialysis Device (Opens new broswer window)
Mark G. Qian, Susan Chen, Debra Liao, Tai-Nang Huang*, Jing-Tao Wu, and Frank Lee DMPK,
DMPK/Drug Safety & Disposition, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA
*Linden Bioscience, Lexington, MA, USA

A Practical Method for Measuring Free Drug Concentration in Whole Blood Using an Equilibrium Dialysis Method (Opens new broswer window)
Susan Chen, Ji Zhang, Tai-Nang Huang1, Jing-Tao Wu, Frank W. Lee, and Mark G. Qian DMPK,
1 DMPK/Drug Safety & Disposition, and Process Technology, Millennium Pharmaceuticals, Inc. 40 Landsdowne Street, Cambridge, MA 02139 USA
2 Linden Bioscience, 35A Cabot Road, Woburn, MA 01801, USA

High Throughput Plasma Protein Binding Assay Using Rapid Equilibrium Dialysis (RED) Device – Faster PPB Assay Results Using a 2 Hour Protocol (Opens new broswer window)
Mark G. Qian, Tai-Nang Huang1, Susan Chen, Ji Zhang, Cindy Xia, Chuang Lu, Jing-Tao Wu, and Frank W. Lee
Millennium Pharmaceuticals, Inc. 40 Landsdowne Street, Cambridge, MA 02139 USA
2 Linden Bioscience, 35A Cabot Road, Woburn, MA 01801, USA

Assessment of the Rapid Equilibrium Dialysis (RED) device for the determination of plasma protein binding (PPB). (Opens new broswer window)
Steve Harris, Jonathan Duckworth, Katie Critchell, Charlie Malloy, Christine Tyman and Mark Savage.
Pfizer Pharmacokinetics, Dynamics and Metabolism Department, PGRD, Sandwich, Kent , UK

High Throughput Protein Binding Using Rapid Equilibrium Dialysis (Opens new broswer window)
Kevin L. Cook, Dennis M. Kalamaridis, Jose Silva, Jie Chen, Carlo Sensenhauser, Vangala Subrahmanyam, and H. K. Lim
DMPK, GPCD, Johnson & Johnson PRD, Raritan, NJ

Development & Validation of Rapid Equilibrium Dialysis for the Measurement of Plasma Protein Binding (Opens new broswer window)
Rachel Jones, Bindi Sohal, Nigel Waters, Gareth Williams
Metabolism & Pharmacokinetics, Novartis Institutes for Biomedical Research, Horsham, UK

References:

1. Waters, N.J., et al. (2008). Validation of a rapid equilibrium dialysis approach for the measurement of plasma protein binding. J Pharm Sci 97(10): 4586-95.
2. Brouwer, E.J., et al. (2000). Measurement of fraction unbound paclitaxel in human plasma. Drug Metab Disposition 28(10): 1141-5.
3. Brunton, L., et al. Goodman and Gilman's Pharmacological Basis of Therapeutics. McGraw Hill Publishing: New York, 2005.
4. Clausen, J. and Bickel, M. (1993). Prediction of drug distribution in distribution dialysis and in vivo from binding to tissue and blood. J Pharm Sci 82: 345-9.
5. Sonnichsen, D. and Relling, M. (1994). Clinical pharmacokinetics of paclitaxel. Clin Pharmacokinet 27: 256-69.
6. Steele, W., et al. (1983). The protein binding of vinblastrine in the serum of normal subjects and patients with Hodgkin's disease. Eur J Clin Pharmacol 24: 683-7.

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