Brad Larson

Automation of Cell-Based Drug Absorption Assays in 96-Well Format Using Permeable Support Systems

Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound’s absorption characteristics.

Automated High-Throughput Purification of PCR Products Using Wizard® MagneSil™ Paramagnetic Particles

Here we describe a reagent system and robotic methods for the purification of PCR(a) fragments from other contaminating amplification reaction components. The methods use the MagneSil™ paramagnetic particle chemistry(b) to isolate double stranded DNA fragments from 150bp to 23kbp. Purified fragments are eluted in water ready for downstream applications such as cloning, fluorescent DNA sequencing and microarray printing. This method has been adapted to a number of liquid handling robotic platforms, including the Biomek® FX and Biomek® 2000 Laboratory Automation Workstations, in both 96 and 384-well formats.

Automated High Throughput Purification of BigDye™ Terminator Fluorescent DNA Sequencing Reactions Using Wizard™ MagneSil™ Paramagnetic Particles

We describe a reagent system and robotic methods for the purification of BigDye™ Terminator sequencing reactions prior to automated fluorescent sequence analysis. The methods use MagneSil™ paramagn...

Automated Luminescence-Based Cytochrome P450 Profiling Using a Simple, Elegant Robotic Platform

The determination of inhibitory effects that lead compounds have on cytochrome P450 (CYP) ezymes is an important part of todays drug discovery process. Assays can be performed early in the discovery process to predict adverse drug—drug interactions caused by CYP inhibition and to minimize the costs associated with terminating candidates in late stage development or worse, removing a drug from the market after launch. For early discovery work, testing substantial numbers of compounds is desirable, thus automated “mix and read” assays are beneficial. Here, we demonstrate the automation of the CYP profiling process using a simple, yet robust robotic platform. Compound titration, as well as transfer of compounds and assay components was performed by the same automated pipetting system. IC50s of small molecule drugs were determined using recombinant CYP enzymes, CYP3A4, −2C9, and −2D6 and luminogenic substrates specific to each. Compounds were profiled against all three enzymes on the same 384-well assay plate.