Hepatic uptake, first-pass metabolism, and biliary excretion are critical parameters in determining the success or failure of a drug candidate. In vitro methods evaluating the hepatic metabolism and induction potential of drug candidates have been extensively utilized.  More recently, the roles of hepatic uptake, biliary excretion, biliary clearance and intracellular concentration of drug candidates are being evaluated. Because the number of hepatic transporters may equal or exceed the number of metabolic enzymes, an in vitro technology expressing the full complement of hepatic uptake and efflux transporters, localized and functioning similar to in vivo, is critical for evaluation of drug candidates. B-CLEAR®, Qualyst’s patented technology, is used to answer questions on hepatic uptake, biliary excretion, and intracellular concentration determination. Automation improves the cost, labor, and time efficiencies in B-CLEAR® experiments, and automation can enable researchers to conduct secondary or small-scale screens in drug discovery and development. This application note describes a protocol to automate studies that evaluate the hepatobiliary disposition (uptake, efflux, and biliary clearance) of drug candidates using Qualyst B-CLEAR® technology on a MICROLAB STAR (Figure 1) from Hamilton Robotics.


Predictions of the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of compounds in pharmaceutical development are essential aspects of the drug discovery and drug development processes. Sandwich-cultured hepatocytes (SCH) (Figure 2) form extensive, functional bile canalicular networks and maintain the expression, localization, and function of uptake and efflux transporters relative to hepatocytes in vivo.1 These canalicular networks, or bile pockets, are isolated from the media by tight junctions between cells. In the presence of buffer containing calcium, [Plus (+) Buffer], the integrity of the tight junctions is maintained.

However, in the presence of buffer not containing calcium, [Minus (-) Buffer], the tight junctions open and release the content of the bile pockets into the media. B-CLEAR® technology (US Patent Nos. 6,780,580; 7,604,934; 7,682,781) uses the modulation of tight junctions in SCH to predict the in vivo hepatobiliary disposition (hepatic uptake, biliary excretion, and biliary clearance), transporter-based hepatic drug-drug interactions, and potential drug-induced hepatotoxicity.2,3,4  The combination of in vivo – in vitro correlation in biliary clearance (Clbiliary) (Figure 3) and accurate prediction of intracellular concentration, makes B-CLEAR® an excellent methodology for predicting hepatic transporter-based drug-drug interactions.5,6,7

Until recently, B-CLEAR® experiments were exclusively performed manually in 6- and 24-well formats. B-CLEAR® experiments are complex in nature, involving parallel timed incubations in Plus (+) and Minus (-) Buffers, and may include multiple substrates at varying concentrations in the presence or absence of potential inhibitors.

Hamilton Company provides world class automation solutions for a variety of applications with increased robustness, flexibility, precision, and accuracy using its unique Compressed O-Ring Expansion (CO-RE) technology. This application note demonstrates the automation capabilities of the Hamilton MICROLAB STAR and how automation can be used to execute B-CLEAR® experiments with a more reproducible and less labor intensive approach, reducing potential operator error and fatigue in complex studies and facilitating technology transfer.


Features and

Automated and manual 24-well B-CLEAR® studies were performed in parallel using d8-taurocholic acid (d8-TCA), a substrate historically used as a control compound in SCH experiments and evaluations. Results from three independent studies, with triplicate wells for each condition, were compared and demonstrated no statistically significant difference using the paired t-test (p<0.05)  in accumulation (cells + bile or cells), BEI, or Clbiliary for d8-TCA between studies conducted either by the MICROLAB STAR or manually. 8SCRH are derived from primary cells and potential differences in lot-to-lot activity are observed in this set of data and are consistent between automated and manual studies.


The process of automating B-CLEAR® technology with the Hamilton MICROLAB STAR has been shown to be a successful alternative to the manual approach. This automated platform is now available for the B-CLEAR® technology, providing a useful tool in drug discovery and development to study the behavior of compounds in the liver. The automation of B-CLEAR® was achieved with Hamilton Robotics highly customizable and flexible automation platforms that can enable a variety of research applications resulting in high reproducibility, robustness, throughput, precision, and accuracy.


Automated Dispenser

The MICROLAB STAR Workstation is a small, flexible, automated robotic workstation that can be adapted to perform a variety of applications. On- or off-deck peripheral devices such as microplate heating/cooling modules, shakers, and vacuum systems can be integrated as needed for the application. The MICROLAB STAR platform uses air displacement technology, which offers increased pipetting accuracy and repeatability while eliminating sample contamination or dilution effects commonly associated with fluid-based systems.

The STAR workstation can be configured with multiple arms, each arm housing multiple pipetting channels or labware gripping options.  Pipetting channels and labware grippers move independently for improved efficiency, and support the use of a wide range of labware.  An autoload option provides real-time barcode tracking of samples, labware, racks and carriers as they are loaded onto the deck. All workstation functions and integrated third-party devices can be controlled by Hamilton Robototics VENUS ONE software. The workstation is built with some unique MICROLAB features such as Anti-Droplet Control, TADM technology, CO-RE technology, capacitative and pressurizing liquid level sensing.

Barcode Tracking 
When the Autoload option is used, the barcodes of all tips, labware, and carriers can be read as they are loaded, confirming the correct items are loaded in the correct location.  A record of all loaded items can be stored and used to control the pipetting process.  Barcode data sets can be compared to worklists from LIMS, exported as data files, or printed. For controlled clinical applications, barcode tracking also provides tracking and record features for samples.

Labware Sensing 
The STAR can monitor and record the hysteresis on the z-drive (up/down) of the pipetting channels.  This information can be used to determine if labware is present or not prior to pipetting, further confirming the method is executed correctly.

Liquid Level Sensing
The STAR pipetting channels are equipped with capacitive and pressure level sensing and recording functions and can be used to check or confirm the liquid level in an individual cell prior to or after pipetting.

Sample Tracking 
The MICROLAB STAR’s VENUS ONE software offers seamless sample tracking over single runs, multiple steps or even multiple runs.


Application note

For Sales and Product information, contact 800-648-5950