A rapid equilibrium dialysis (RED) device that transforms technologies for performing plasma protein binding assays.

The Thermo Scientific Pierce RED Device for Rapid Equilibrium Dialysis was developed in close association with the pharmaceutical industry to provide the easiest, fastest and most reliable system for performing plasma protein-binding assays, which comprise a critical step in drug development.

The RED Device is an apparatus for performing equilibrium dialysis experiments in a high throughput, automation-compatible format. The device consists of disposable inserts and a base plate formatted to a standard microplate footprint. The RED Device has been extensively validated for plasma-binding assays producing results consistent with those reported in the literature. The Rapid Equilibrium Dialysis (RED) Device offers significant improvements in automation, time requirements, versatility and product reliability compared to other commercially available systems.

Highlights of the Rapid Equilibrium Dialysis Device:

• Easy and ready to use – disposable tubes require no presoaking, assembly or specialized equipment
• Designed for speed – the high surface-to-volume ratio of the insert design enables equilibrium to be reached in as few as 100 minutes with vigorous agitation or in three to four hours with 200rpm agitation
• Automation-compatible – designed on a standard 96-well plate template suitable for automated liquid handlers
• Flexible and scalable – perform any number of assays (1 to 48 assays per plate) without wasting the entire 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
• Quality-tested – each lot of inserts is functionally tested in a protein-binding assay for guaranteed performance

Features of the RED Device Inserts:

Each single-use, disposable insert is made of two side-by-side chambers separated by a vertical cylinder of dialysis membrane (8K MWCO) validated for minimal nonspecific binding. This format requires no extensive assembly steps or specialized equipment, and each chamber or well is easily accessible from the top of the insert after insertion in the base plate. Additionally, the high surface-to-volume ratio of the membrane compartment allows rapid dialysis, where equilibrium can be reached in 4 hours with high levels of reproducibility and accuracy. In many cases, experiments can be completed in less than 100 minutes.

Insert Features:

• Disposable: require no presoaking, assembly, or specialized equipment
• Short incubation time: large dialysis surface area accelerates equilibrium
• 8K MWCO membrane: ideal molecular-weight cutoff for protein-drug binding studies
• Membrane composition: regenerated cellulose with low glycerol content as a humectant

Features of the RED Device Base Plates:

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 9mm x 9mm 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.

Base Plate Features:

• Microplate footprint: compatible with automated systems for 96-well plates
• Compartmentalized: eliminates potential for crosstalk or leakage
• PTFE construction: eliminates nonspecific binding and risk of contamination
• Accepts 1 to 48 inserts: run exactly the number of assays needed without waste

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

Additional Information:

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 has been extensively validated for plasma-binding assays producing results consistent with those reported in the literature. For example, when used to measure warfarin binding to plasma (human and rat) proteins at two concentrations of 1µM and 10µM, the RED Device produced results with minimal intra-experimental variability. The Rapid Equilibrium Dialysis (RED) Device offers significant improvements in automation, time requirements, versatility and product reliability compared to other commercially available systems.

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.

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)
by 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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