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Screening with high quality and high throughput

The Early Discovery department of the research-based pharmaceutical company Nycomed GmbH deals with early drug research, from the identification of targets to the generation of lead structures. This process involves high-throughput drug screening using three robotic lines that fulfil different requirement profiles. On behalf of www.bio-pro.de, Michael Statnik talked with Dr. Stefanie Polej about the process of the automated analysis of molecule activities in the Constance-based company.

Dr. Polej, since when has Nycomed been using high-throughput screening?

Nycomed (formerly ALTANA Pharma and Byk-Gulden) has carried out high-throughput screening (HTS) since 1998.

Which systems and machines do you use in your laboratory for the automated search for lead structures?

As far as HTS screening is concerned, we use three different robotic lines which are used according to our requirements. The use also depends on the robots’ strengths and the measurement devices available on the respective robotic line.
One of these robotic lines is used for standard screening, i.e. high-speed and high-throughput screening with more than 100,000 assay points per day. The second system (up to 60,000 assay points per day) is used for enzymatic assays and allows the high-throughput screening of radioactive assays. In addition, we also have a very flexible robotic line that can be used for high-throughput confocal microscopy.
HTS robotic lines for high-throughput screening
Nycomed GmbH uses three different HTS robotic lines for high-throughput screening (Photo: Nycomed GmbH)
The number of actual assay points per day depends, among other things, on the throughput rate of the measurement device: the recording of microscopic images and the time required to do so depends on the exposure parameters. This process is similar to that of a digital camera, with the only difference being that several hundreds of thousands of pictures have to be taken and processed in a single screening campaign. The process requires enormous computer capacities, but also a lot of time. The imaging system of our robotic line allows us to process over 65,000 assay points per day.

Can you give me some details about the robotic lines and the screening processes?

Our most modern screening system is equipped with a central conveyor belt that transports the microtitre plates. At the individual workstations, robotic arms take the assay plates and feed them into the transport process between the individual devices. Several stations can be served simultaneously as long as the conveyor belt has free places available. This guarantees maximum capacity utilisation and makes the robotic line extraordinarily fast. In contrast, our highly flexible robotic line is equipped with a mobile robotic arm that is mounted onto a guide rail. Since this arm has to feed the pipetting, incubation and measurement stations located on the left and right, this type of set up reduces the throughput speed.
Robotic system for pipete handling.
A 384-tip pipetting head transfers substances to 384-well and 1536-well microtitre plates (Photo: Nycomed GmbH)

How many assays can be screened?

In general, the more complex an assay, the greater the probability that the number of potential assay points decreases. For example, if the robot-assisted main screening involves primary cells, the cell culture supplying the cells might turn out to be the limiting factor in this process. The same is true for the incubation time required or the number of additions, e.g., another compound and reagents or additional buffer solutions. This is not so in standard screening processes, which means that complex assays require a much high number of robotic runs. If a pharmacologically important, but complex and time-consuming model is chosen, then a slower speed is selected. This leads to higher data quality because additional important parameters can be tested. In general, the throughput has increased considerably over the last few years through the miniaturisation of the assays to as little as five to eight microlitres per assay point.
View into a lab with a screening facility in the background.
Nycomed’s compact, modular screening facility, which also involves the transportation of microtitre plates on a rail system (Photo: Nycomed GmbH)

Which drugs are tested for their interaction?

Normally the screening focuses on the functional properties of the targets; this means, pure binding assays are unusual and are only used for drug profiling. The main screening involves all common test formats, including enzyme tests, cellular tests, including reporter gene-based as well ion channel tests. It is important that the targets that are to be screened can be transferred into a measurable detection format for which we use common methods, i.e. microtitre plate readers for measuring absorption, different fluorescence methods, luminescence, confocal microscopy up to 1536-well formats, but also radioactive screening. In general, our high-throughput screening involves 384-well or 1536-well microtitre plates.

Why do you carry out secondary screening? Can this be used to clarify additional questions, for example those on the solubility of substances or whether the substances bind to other parts of the cells?

Secondary screening, which is also fully automated, involves the dose-dependent screening of drug effects. We collect information on whether the hits of the primary screening have undesired side effects on related targets, which would make them unsuitable for further processing. Routine investigations also collect information on initial ADME category parameters such as absorption, distribution, metabolism, elimination. Hit evaluation also includes solubility and permeability screening as well as data from toxicological assays. This type of screening also assesses the metabolic stability of drugs and their interaction with cytochromes in order to guarantee the highest possible quality of lead structures and allow medical chemists to make initial prioritisations on the basis of a far more complex drug profile than was possible a few years ago. All assays are carried out under standard conditions and are highly automated. Suitable quality control measures provide information on the course of the screening and the results will only be made available upon the positive evaluation of the process.

Are there situations that require manual experiments rather than automation?

This is the case in more complex test systems, which cannot be transferred into a microtitre plate-based, robot-suitable format and which must be carried out with far smaller amounts of reagents and test substances. The decision on whether automated or manual screening is used depends on a variety of factors.
Background: Dr. Stefanie Polej studied biochemistry at the University and Medical School in Hanover, Germany. She did her doctorate at the Max Planck Institute for Biophysics in Frankfurt and then worked in pharmaceutical research as the laboratory head at Bayer AG’s Institute of Molecular Screening Technology before accepting a position at ALTANA Pharma. Following the acquisition of ALTANA through Nycomed, Dr. Polej now works in the company’s Early Discovery department.
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