Efficient Sample Logistics: From the Chemist to the Assay Plate and Beyond

Introduction 

There is increased demand within the drug discovery industry to reduce the turnaround time from when a compound is synthesized to when target, off target, and ADME assay data become available to medicinal chemistry project teams.1, 2 Consequently, there is a need for the reliable and rapid flow of samples from the chemistry laboratories into a variety of assay-ready plates and to the corporate sample collections for screening and counterscreening. For some time, Terlings Park Neuroscience Research site of Merck Sharp & Dohme has had an efficient process for transferring 10mM DMSO solutions of recently synthesized compounds from our on-site medicinal chemistry teams to the central Merck & Co. Compound Management Group in Rahway, NJ, for subsequent global distribution. More than 80% of new samples are captured and shipped in a timely manner to enable further distribution into the screening collections and for use in other projects.

This high-capture rate of new samples is very important because it increases the diversity of the available collection, increases the representation of compounds which have been targeted to neurological disorders, and ensures that the company gets the maximum value from these in-house compounds. Pharmaceutical R & D organizations may spend large amounts of money to increase their sample collection without making the best use of their internal resources—some estimate that less than 20% of in-house prepared compounds may make it into some company compound sets.3

An efficient process of sample collection, manual storage, and reformatting into assay plates has been part of our group's workflow for some time. In 2003, it was decided that we should automate the storage and retrieval of these samples to help further streamline the workflow and reduce the time required to locate and make available a requested sample. This new process (Fig. 1a), and the technology underlying it, is described below.

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Figure 1. (a) Workflow of samples, (b) handheld bar code scanner used to record sample collection data, and (c) Biomek FX used to transfer samples from vials to 2D bar-coded tubes.

Collection of the samples from the chemist and reformatting 

When new chemical entities are prepared within medicinal chemistry, they are registered using proprietary software and allocated a Merck corporate identifier in the format L-XXXXXXXXX-000A001. The chemist then accurately weighs out a proportion of the solid (5–10mg) and prepares stock solutions at 10mM in neat DMSO. From this stock are prepared the assay sample in a 4-ml vial and a sample in a 20-ml vial to be shipped to the central Compound Management Group in Rahway, NJ. Both vials are identified by a bar code label that encapsulates the Merck corporate identifier, the chemist's name, sample volume, and concentration. The assay sample is also coded to identify the project and assay for which it is initially destined—this is via a colored dot on the lid. The samples are collected during a daily collection round of the chemistry laboratories by a member of the dedicated compound management team; compound vials are scanned using a portable handheld bar code reader (Symbol Pocket PC) that records the laboratory, the L-number, and the primary target assay at the point of collection (Fig. 1b). This information is transferred to an Access database held on the Merck network. This uses MCL-Client, a Windows-based software for controlling handheld and mobile devises written in MCL-code scripting language, and MCL-Link, a communication program that allows point-to-point communication between a device and the host computer via a docking station, provided by Peak Technologies UK (Ascot UK, http://www.peakeurope.com).

The samples destined for the corporate Compound Management Group are racked in groups of 80 ready for shipping, two racks at a time (160 samples), and then sent by air freight in dry ice to Rahway, NJ. This process has become more difficult in recent years with the increased bureaucracy on compound imports to the United States. A specialized courier company, Biocair International Ltd (Cambridge, UK), is used to reduce the risk of the samples being held up in customs and of thawing. The samples are shipped in dry ice for two reasons: firstly, to reduce any degradation of the compound in solution, and secondly, to avoid spills (although a specially designed cap with an insert in the lid is used on the 20-ml vials, it is still possible that leakage occurs around the lid during the transit if the sample is not frozen). Once the samples reach Rahway, they are processed and become available to both HTS screening collections and substructure/similarity screening around the Merck global R & D network. The high success rate of >80% of new chemical entities from the medicinal chemistry teams at Terlings Park reaching the corporate collection can be attributed to a number of policies. These include targets being set in individual chemists' objectives for submitting compounds to the collection; the realization by the chemistry teams that they need these types of structures that are targeting neurological disorders in the sets used to screen against new neuroscience targets; an encouraging management culture that understands this is the correct way to get the best out of their synthetic endeavors; and the existence of this group that provides the infrastructure to easily and conveniently process these samples.

Processing the assay samples 

The 4-ml vial containing the 10mM DMSO solution destined for project assays at Terlings Park is processed by the compound management team. The first step in the process is to transfer the sample from the bar-coded vial into a Matrix 1.4-ml 2D coded V-bottomed tube along with the tracking of the L-number and the 2D bar code. Physical transfer is carried out using a twin pod Beckman Biomek FX; an eight-span pod with liquid sensing disposable tips is used for this process. The use of disposable tips reduces the risk of cross contamination and removes the need to dispose of large amounts of aqueous DMSO waste, which would be needed to wash fixed tips (Fig. 1c). For the 4-ml vials to be held accurately on the deck of the FX, initially a number of custom racks were fabricated in-house, however, the final solution was to use a 24-well plate cell culture plate from Corning (Cat. number 3526). Generally, 200μl of a 10mM solution is transferred, although the transfer program can deal with a variety of volumes and concentrations.

Process supporting database and software 

A key part of the process is the in-house build database Autopicker, which associates the L-number with the 2D bar code on the tube. The 2D bar code information is only used within the confines of this software and the tube storage tracking software; outside of this, all references to the sample are by the L-number. Autopicker is a Java Server Page application running on an Apache Tomcat server backed by an Oracle 9i database. The application was built using an in-house Model-View-Controller architecture similar to the Jakarta Struts framework. To make the initial link between the L-number and the 2D bar code in the database during the loading process, a rack of tubes is scanned using a commercially available scanner and decoder software. The output is a text file containing both the decoded 2D bar code and the tube location in the rack; this is then cross-referenced against a rack ID. The input page of the Autopicker software allows for the input of this text file, which automatically fills the left-hand panel of the input form (Fig. 2) allowing the L-numbers to be scanned in directly from the bar code label on the vials into the right-hand panel using a handheld keyboard wedge bar code scanner (Intermec Sabre 1551). At this point, both the concentration and the load volume can be entered, and by using the “submit” button, the link is made between the 2D tube bar code and the L-number (Fig. 2).

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Figure 2. Screenshot of the input page of the Autopicker database built in-house.

Which automated tube store to use? 

Three key factors were used to decide on the tube storage system to purchase: (a) storage density, that is, highest number of tubes in the smallest footprint, (b) the ability to store and retrieve multiple racks of tubes without human intervention, and (c) easy use of the software. Based on these key criteria, we chose ComPOUND from TTP LabTech Limited (Melbourn, UK). ComPOUND (http://www.ttplabtech.com/compound/index.htm) can hold up to 100,000 1.4-ml tubes, which are stored in a controlled environment (nitrogen atmosphere from ambient to −20°C). We run our system at 5°C.4 The primary unit is a ComPOUND storage module (Fig. 3a), which can operate either alone or with other units when larger storage capacity or higher throughput is required. The ComPOUND software provides an easy and foolproof interface between the module(s) and any existing LIMS or Compound Management Systems. ComPANION is a remote arrayer unit that assembles racks up to 15m away from the ComPOUND module: it can easily be automated as part of a secondary robotic platform.5 In our case, it is integrated with a Hamilton Swap and three hotels (ComMOTION) allowing for 24 tube racks of 96 wells to be handled without human intervention (Fig. 3a,b).

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Figure 3. (a) ComPOUND, (b) ComMOTION, and (c) diagram of the inner working parts of ComPOUND.

The ComPOUND/ComMOTION combination is controlled by a single PC with a touch screen. To enter samples, the user configures the very intuitive visual interface (Fig. 4a,b) with the number of tube racks to be stored and their location within the three hotels, and then touches the “start” button. The system has a built-in Hamilton Swap robot arm moving to each hotel in turn with the robot fingers locating two pegs on each hotel to ensure that the correct hotels are in place before the storage process starts. The Hamilton Swap then transfers the rack of tubes to be stored onto the input tray on the ComPANION. As the tray moves into position, a height sensor ensures that no tubes are sticking up in the rack. If they are, it rejects the rack and returns it back to the hotel and moves to the next rack to be stored. Once in place, an integral bar code reader scans the tube rack and then, using compressed air, the individual tubes are transferred from the tube rack in ComPANION to the input turntable in ComPOUND via a transparent transfer tube. Once inside the turntable, a 2D bar code reader scans the tube and logs the tube ID with a location ID indicating where the tube will be stored. The tube is then delivered to the correct location in one of 26 carousels; each contains 3850 storage holes for tubes, tapered to match the tubes, arranged in 31 concentric circular rings. Each carousel also has a row of large “through holes” or “delivery holes” across its diameter, which are used to move tubes to storage holes. There is one through hole for each ring of storage holes. When all the carousels are parked, the through holes line up with each other to make a series of chimneys, from top to bottom of the carousel stack through which tubes can pass (Fig. 3c). If the 2D bar code reader fails to read the code on a tube (which is very rare), the tube is stored but flagged as a “Failed to read bar code of vial during store”, and the software allows the user to retrieve these samples for reprocessing.

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Figure 4. Screenshots of the ComMOTION software (a) configuration page used to indicate the locations of tube racks to be stored and the empty tube racks ready for retrievals and (b) in running mode.

Storage and retrieval of tubes for compound 

Using the touch screen, the operator can highlight hotel locations with tube racks containing the tubes to be stored, and then select the “start” button (Fig. 4a). The system will automatically process all of the racks with tubes to be stored. These can be full tube racks, partially filled tube racks, or a mixture of both. The ComPANION scans across the complete rack looking for tubes to store. The system takes around 8min to store 96 tubes.

To retrieve samples to be plated for assays, the operator places an order in the Autopicker database; the starting point for this is a list of L-numbers that has been requested for an assay or counterscreening. An order has to be initiated within the Autopicker software to convert a list of L-numbers into an order containing 2D bar code information that the ComPOUND software can use. This is carried out by a member of the compound management team. Because the software is Web-based, the order can be conveniently processed from any computer with Web access.

A list of L-numbers from any source can be copied into the order page of Autopicker (Fig. 5a); at this stage, the operator can also pick the required tube layout for the output tube rack and the volume to be removed. The software tracks volume throughout the process and will not allow an operator to order a tube that has less than 10μl remaining. The tube rack layout is selected using a visual plate map that allows for the selection of individual locations, rows, columns, or the whole plate. The operator can save these plate layouts as templates and recall the same plate layout for future orders. When a plate layout has fewer slots than the number of ordered samples, the system will place the excess samples on to the next plate, and so on until the last compound in the order list has been located into a tube rack and location.

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Figure 5. Screenshots of the Autopicker software (a) order page showing selected tube positions for retrieval and (b) tube location map available for data export.

Once the order is submitted, the database provides a visual plate map (Fig. 5b) containing the 2D identifier and the L-number; this information can be exported to Excel for use in the assay scientist's data analysis software. At this stage, the database can automatically send the order text file to ComPOUND, which only contains the 2D bar code information that the ComPOUND needs to locate the sample. The order is placed in the “Orders” folder on the ComPOUND PC, which is shared on the internal network. The format of this text file (Fig. 6) is such that it contains both the final well location within the output tube rack and the tube 2D bar code. This is known as Fixed Delivery Retrieving mode, that is, each tube has a prespecified location within the tube rack and will not be placed in a random slot. The ComPOUND software does not need tube locations; 2D bar codes are sufficient to retrieve the samples in an order that is most efficient for the system and provide an order report that lists the locations. This is known as Random Delivery Retrieving mode, that is, the samples will not be retrieved in the order they are listed on the order file, instead they will be retrieved in a random order corresponding to the minimum number of carousel movements. Because we want to specify the location for an L-number routinely, we do not typically operate ComPOUND in this mode. The order file has the filename format assay1.order and is recognized by the ComPOUND software as a new order waiting to be processed. Once the order is processed, the file extension is changed to assay1.order.report, and as such is no longer recognized as an order file.

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Figure 6. An example of a ComPOUND orders file.

The ComPOUND software continually scans the orders folder for new orders, and upon detecting one schedules the order in the ComPOUND calendar in a manner similar to that in an Outlook calendar. If the ComPOUND/ComMOTION system is set to “start” mode rather than “configure” mode, and sufficient empty bar-coded tube racks have been loaded, then the order will start to run without further human intervention. If the system is in “configure” mode (Fig. 4a), the system can be configured with empty racks; racks to store and completed racks can also be removed. Once there are racks set to store and empty racks in place, the Configure button is touched and changes to “Start” allowing the system to store the tubes in the racks highlighted as store, and subsequently retrieves any outstanding orders. When an order has been completed and the racks of tubes are in the hotels, selecting a “retrieve” rack on the screen displays the order details including contents, order name, and rack bar code in the right-hand window. This aids the operator when removing orders from the system. Once the tube racks have been removed from the hotels, they are transferred to a Velocity 11 (Palo Alto, CA) VSPIN centrifuge configured within a BenchCell 2X stacker system (Fig. 7a). The tube racks are spun at 1260 rpm for 7s to remove any sample that may have been transferred to the tube cap during the retrieval process and to minimize sample waste. The caps are then removed from tubes manually; tubes are then ready for use in the plating process.

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Figure 7. (a) Velocity 11 VSpin and BenchCell integration used to spin tubes before decapping and (b) Biomek FX transferring samples from 2D bar-coded tubes into 384- and 96-well assay plates.

Transfer of samples from the tube to the assay plate 

The tube racks are transferred onto a twin pod Beckman Biomek FX, using the eight-span probe with liquid sensing disposable tips (Fig. 7b), and by running predetermined assay-specific programs either 384-well or 96-well assay plates are prepared; the plates have previously been bar coded offline. In general, each compound is transferred to one or two wells in the plate in preparation for a 10-point serial dilution protocol, which is carried out in 100% DMSO. The plates are then sealed using either a heat sealer, or with adhesive seals, and delivered to the assay team scientist. The tubes are capped with fresh caps either using a Matrix automated capper, or by hand, and returned to ComPOUND using the same procedure as described above.

Conclusions 

A streamlined workflow has been established within our compound management team to facilitate reliable and efficient delivery of DMSO solutions of new chemical entities to primary project assays and the Merck global screening collections. At the center of the process for in-house assays is the use of 2D bar-coded tubes in combination with ComPOUND/ComMOTION software, a Beckman BioMek FX, and a database built in-house. This arrangement has allowed us to develop an efficient process to store and retrieve compound samples, prepare assay-ready plates, and re-store samples. The main labor savings have resulted from the speed and ease with which samples can be retrieved from the automated store compared to a manual store. In addition, important efficiency and productivity improvements have resulted from the accuracy of the compound identification and retrieval using the database in combination with ComPOUND scanning the 2D bar code on both entry and exit. This minimizes any possibilities of erroneous samples being tested or important compounds being accidentally eliminated from primary assay plates. The hardware and software components of this process have proven to be very reliable in routine use within our sample logistics and assay screening workflow.