Abstract
Whereas industry was using 96 well microplates until the beginning of the 1990s, the sample density has meanwhile increased sixteen-fold to 1536 well microplates. In large pharmaceutical companies, a similar trend to that experienced in the semiconductor industry, is seen whereby the number of transistor functions on the same surface area quadruples every three years. However, a sample density of 1536 wells represents a major challenge for the pharmaceutical companies in their search for active constituents — both financially and technically. The acquisition of new equipment is imperative, and considerable investment in work and time are necessary to ensure smooth running with 1536 well plates. The result of such efforts is of course fantastic. Dr. Martin Bechem, Head of Molecular Screening Technology at Bayer AG, Germany, expects to achieve up to 200,000 active constituent tests per day, depending on the test system used. Dr. Bechem's research group recognized the possibilities of the 1536 well plates at an early stage and, in co-operation with GREINER LABORTECHNIK, was instrumental in their development for practical application. The result of this distinctly successful cooperation was the first commercially available 1536 well microplate, which satisfied all the demands of automated high throughput systems. A satisfying by-product was also the submission of German, European and American patent applications for the production of microplates with clear bottoms, without the use of disturbing adhesives or solvents. The availability of such μClear® plates will have a considerable influence on new test methods and the spread of further detection techniques, since it means that all plate formats can also be measured from below. Other companies are also opting for the 1536 well format, such as Aventis (formerly Hoechst Marion Roussell), Bristol-Myers Squibb, Glaxo Wellcome, Hoffmann LaRoche, and Novartis.
In order to ensure smooth running of the tests, in addition to the latest automation methods, long-term and reliable support of the 1536 well plate format is imperative. GREINER LABORTECHNIK was the driving force behind this format from the very beginning and repeatedly achieved new technological advances at an early stage. The latest baby of the 1536 well family is a reagent storage plate (deep well plate) made of high clarity polypropylene with a well volume of 20 μL. The visitors at LabAutomation were understandably astounded. An original comment from one of our customers: “None of your competitors has mentally gone for it to such a degree, and you've already achieved it.” The close co-operation with numerous users has not only led to the creation of a broad product range, from complete white and black plates to μClear® microplates, but also to constant improvements in quality. All plate types are available with a variety of different surface treatments, e.g., for tissue culture or immunoassay applications.
Greiner will emphasize its leading position in high density plates based on a unique molding technology with an increasing variety of plates. Several new products are under construction right now, like the 1536 well HiBase plate, glass bottom plates (96, 384, 1536 well), and a generation 2 of the 384 SV plates. But there are already requests which go beyond the features of the new 1536 well generation 3 plates. Some of them are directly related to the launch of new robots, stackers, 384-well dispensing heads, and detection techniques. To make these even more advanced plates available, we have already started to work on the 4th generation of a 1536 well plate. ▪
A contribution by Dr. Rainer Heller and Dr. Günther Knebel
Training and customisation were big issues for Prolysis Limited when they decided to buy their first big, automated assay system. Prolysis uses its new TECAN system to develop high throughput screening assays for the pharmaceutical industry. The assays are used to identify and optimise antimicrobial lead compounds.
When Dr. David Foulger from Prolysis Limited decided to automate assays in his laboratory, he turned to TECAN for this flexibility.
Prolysis Limited was formed by Professor Jeff Errington, from the Sir William Dunn School of Pathology, at the University of Oxford. The University and Oxford Molecular Limited, a discovery research company, are major shareholders in Prolysis Limited.
As part of Oxford Molecular's Collaborative Discovery programme, Prolysis develops and operates high throughput screening assays for the identification and optimisation of antimicrobial lead compounds. Prolysis can either perform the assays on behalf of the pharmaceutical companies or licence a developed assay for them to run themselves on robotic systems.
The assays are primarily searching for genes involved in cell division. Some are general, looking for any inhibitors of cell division whereas others are specific for particular division proteins.
Other users in and around the University recommended TECAN to Dr. Foulger. Having had limited previous experience of using laboratory robots, it was important from the outset for him to have excellent technical support.
Before even purchasing the system, he gave TECAN the existing assay protocols to work on. The team was able to simulate the assays using TECAN FACTS (Flexible Assay Composer Task Scheduler), software and gave Dr. Foulger approximate timings of the assays, including time allowed for the incubation stages.
The more complicated sections of the assays were set up on TECAN modules in the TECAN UK workshop so that he could see them up and running and make any necessary adjustments before the system was installed. This level of preparation reassured Dr. Foulger at a very early stage that the TECAN system could actually do what he needed it to do, an important consideration when investing a lot of time and money.
The workstation that TECAN configured for him included a GENESIS Robotic Sample Processor (RSP), 200 with a pipetting arm, tip washer and a Robotic Manipulator (RoMa) arm which transfers the plates between the worktable, three incubators, plate hotels and a SPECTRAFLUOR microplate reader.
The assays were originally performed manually using 8-channel pipettes so that the introduction of the TECAN Workstation has meant a ten-fold increase in the rate of sample throughput. It can currently process 15 plates per run, with time for multiple runs per day.
The GENESIS RSP takes samples from a chemical library and makes serial dilutions down a 96-well plate to varying concentrations depending on the assay requirements. If necessary, it will also create ‘daughter’ plates from this original ‘mother’ plate. These pipetting stages were some of those re-created in the TECAN UK workshop early on in the development of Dr. Foulger's system so that they were absolutely right when it came to installation.
After a few days initial training when the system was installed, Dr. Foulger found it helpful to be able to use the system for a couple of months before attending a TECAN training course. At this stage he was much better prepared to ask questions relevant to his own system and found the extra tuition very useful. He attended a three-day residential course covering basic LOGIC software as well as FACTS, with a whole afternoon dedicated to questions and answers.
Not surprisingly, as work developed over several months, some stages of the assay protocols needed to change. Dr. Foulger could, in many cases, adapt the software himself, with technical support from TECAN at the end of the telephone when needed. Some problems were more complicated.
One such problem was to do with two consecutive readings taken at different wavelengths by the SPECTRAFLUOR. Originally, the RoMa arm moved the plates back to the worktable between the readings but the TECAN team reconfigured the software to stop this happening.
Dr. Foulger also experienced the common problem of sample evaporation due to relatively long incubation times at 37°C. He decided to change to plates with lids, which brought its own problems because the lids made the plates higher and the software settings did not allow for removal of lids. TECAN reprogrammed the software so that the RoMa arm removed the lids during pipetting stages, returned them to the plate hotels and then replaced them for incubation stages.
Another common problem encountered by several users of automated systems has been settling of samples. One particular customer used a Synchron magnetic flea in a pool of bacteria to prevent settling. The TECAN hardware was reconfigured with a light sensor to keep the magnetic flea stirring at appropriate times but to stop them during pipetting stages.
Many TECAN users working in the drug discovery field are currently adapting TECAN systems for cell permeability studies. The TECAN system uses special plates which contain inserted cups. These have a permeable membrane on which a mono-layer of cells is grown. The cells need to be kept continually moist with enriched media.
A drug or unknown substance is then dosed into the top well and, after a designated time, samples are taken from the top and bottom wells. From these measurements, you can evaluate how efficiently that substance is transported actively across the membrane, and assess its suitability as a drug. ▪
TECAN SUPPORT
Looking back on the last two years, David Foulger generously praises the TECAN team for their total support and understanding. “It made all the difference,” he says, “and has encouraged me to explore the flexibility of the system, and experiment with the new methods that will help to expand our business.” TECAN AG, Feldbachstrasse 80, 8634 Hombrechtikon, Switzerland
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