Laboratory automation – automated sample purification

An alternative that is used primarily in the purification of proteins, process monitoring, and downstream processing is chromatographic purification. Parallel chromatography is possible with high throughput by using RoboColumns. These are miniaturized chromatographic columns with different packing materials.

The increasing number of samples to be processed have caused the need for parallelization to process several samples at the same time. Various solutions are available for this today.

Automated solid-phase extraction

Various variants are conceivable for carrying out solid-phase extractions. If a simple gravity-based elution of the samples is not possible, this can be achieved either vacuum-based or by applying overpressure to the columns. With vacuum-based SPE, the liquid is drawn through the cartridges by applying a vacuum. However, problems can arise with parallel SPE methods, as applying and maintaining a uniform vacuum on several columns can be problematic. An alternative is to use overpressure to push the liquids through the SPE cartridges. With the help of overpressure SPE, a higher pressure consistency can be achieved on the columns. Suitable automation and device design with electronic pressure control offer the possibility of providing set pressure values for conditioning, sample transfer, and washing steps, as well as the line pressure for drying before the elution step.

Regarding the level of automation, a distinction can be made between semi- and fully automatic systems. While fully automated systems allow autonomous processing of samples, semi-automated versions require manual process steps. To enable fully automated solid-phase extractions, the devices must have an interface for control by a higher-level system. Furthermore, the selective loading of the column and elution labware by a robot arm or the gripper of a liquid handler must be possible. There are proprietary systems which have a pipetting arm for feeding the samples, standards, etc. However, extraction devices that can be integrated into commercially available pipetting robots and allow direct pipetting access to the labware in the extraction device offer greater flexibility. Such systems are also easier to use for higher automated throughput, as they provide more labware capacity on the liquid handler. The systems should also have options for conditioning and washing the columns while simultaneously draining the liquids into the liquid waste.

A simple variant of the automation of solid-phase extractions is represented by systems that can process individual samples sequentially. Such systems are available from manufacturers such as Gerstel (Mülheim, Germany), LCTech (Obertaufkirchen, Germany), or Gilson (Middleton, WI, USA).

There are two basic types of devices for parallel processing: systems that can process a maximum of eight columns simultaneously; and systems that enable the processing of up to 96 samples in microtiter plate format [5]. An example for systems with limited parallelism is the Solid Phase Extraction System Speedy (Zinsser Analytics, Eschborn, Germany).

Highly parallel systems enable at least 96 samples to be processed in parallel. The systems from Tomtec (Hamden, CT, USA) and amplius (Rostock, Germany) also enable the processing of 384 parallel samples; A maximum number of 1,536 parallel samples is specified for the Quadra 4. A few systems ([MPE]2, Hamilton, Reno, NV, USA; Positive Pressure Unit, amplius, Rostock, Germany) allow a flow limitation of the gas used for pressurization for each SPE column. This ensures that the pressure set for pressurization is always present on each of the channels, regardless of the number of columns installed. In addition, this flow limitation per channel ensures that the pressure for all columns remains stable, even if individual columns run faster than others due to the sample. To detect blockages on the individual columns, the Zephyr G3 SPE workstation (Perkin Elmer, Waltham, MA, USA) is also equipped with an ultrasound-based sensor (PING Non Contact Clog Detection). For the greatest possible flexibility, different column formats, such as microtiter plates or individual columns fitted in adapters with microtiter plate dimensions, should be supported. Columns with different adsorbents are traditionally used in solid-phase extraction. In principle, these can also be used in automated systems. There is currently no standard regarding the dimensions of the labware for solid-phase extraction. Typical column sizes today are 1 mL, 3 mL, and 6 mL. The individual columns are either fitted into racks permanently installed on the liquid handler deck or inserted into mobile racks. The latter variant is usually in microtiter plate format, which ensures that automated systems with robotic grippers can transport the racks with columns, and thus work can also be carried out in automated high-throughput. Such racks must always be adapted to the type of column to be used. Microtiter plates are also used to ensure high parallelism. A distinction is made between solid-phase extraction plates in one piece (molded format) and corresponding plates in a modular format with removable wells. The use of modular plates enables the flexible assembly of the plates with different columns. In this case, the plates can also be equipped with different columns, which is particularly important in the area of method development or can be used for smaller numbers of samples.

Automated solid-phase extractions have been used for the determination of vitamin D in blood samples [6], high-throughput filtration in cannabinoid analysis [7], the determination of dental substances [8], or the extraction of cyclophosphamide from cells and cell supernatants for the determination of active ingredient uptake [9].

Chromatographic sample cleanup using RoboColumns

RoboColumns are miniatures of classic chromatography columns that can be used for the purification, separation, or concentration of compounds. The much smaller RoboColumns have the great advantage that they save material. This applies on the one hand to the column material (stationary phase, etc.), and on the other hand to the sample that is to be separated. They also save time, as separation is completed much faster on the smaller columns. The RoboColumns are used less for the actual separation of a mixture of substances, but rather for the screening of factors that influence chromatography. The basic idea is to test different chromatographic conditions (stationary phase, buffer, etc.) for a substance in parallel to develop an optimal purification process. Such a screening for chromatographic conditions and materials would be too expensive and time-consuming with the classic, larger columns [10].

By using RoboColumns, process development can be carried out with high throughput, which saves the industry time and often also costs. RoboColumns are also suitable for monitoring bioreactors (or fermentors) in which microorganisms or eukaryotic cells are cultivated under almost optimal conditions. Bioreactors are used in sewage treatment plants, biogas plants, in the food industry (e.g., breweries and wineries), and in the pharmaceutical industry. With the help of RoboColumns, these processes can be monitored within the bioreactors without using large sample quantities [11].

The selection of column material (stationary phase, etc.) is now huge and can be customized for the customer. In addition to Atoll (taken over by Repligen Corporation in 2016), other companies such as Cytiva (Marlborough, MA, USA), Bio-Rad (Hercules, CA, USA), Tosoh (Tokyo, Japan), ThermoFisher (Waltham, MA, USA) and Sartorius (Göttingen, Germany) also offer their versions of RoboColumns. RoboColumns are usually supplied in rows of eight individual columns and can be adapted to the 96-well format. The column volumes are typically 50 µL to 600 µL. In addition to the classic RoboColumns, some manufacturers also offer plates in MTP format (with correspondingly smaller column volumes). Different column materials enable the performance of ion exchange, mixed-mode, hydrophobic interaction, and affinity chromatography. In affinity chromatography, the high affinity or tendency to bind between two (bio)molecules is used as a separation principle. Hydrophobic interaction chromatography, on the other hand, is based on the specific arrangement of amino acids in proteins. Compounds can also be separated using ion exchange chromatography based on their charge by using cation or anion exchange resins as the stationary phase. In multimodal chromatography, several of the separation principles mentioned are combined to induce secondary interactions.

Automated chromatography is possible in combination with robotic liquid handlers. The OPUS RoboColumn was originally developed in close collaboration with Tecan (Männedorf, Switzerland) and can be processed automatically on the Freedom EVO series and Fluent systems. Automated protein purification is also possible with the Janus G3 BioTX Pro Workstation and the Janus G3 BioTX Pro Plus Workstation (Perkin Elmer, Waltham, MA, USA). The RoboColumn Unit (amplius, Rostock, Germany) is a system for automated chromatography on the Biomek series NX, FX, i5, and i7 (Beckman Coulter, Indianapolis, IN, USA). Integration into other liquid handling systems is also possible.

Read the articles of the series “Automation in the Laboratory”:

Part 1: Definition, applications, and potential of laboratory automation. 

Part 2: The LUO concept in laboratory automation. 

Part 3: Liquid handling in laboratory automation.

Part 4: Low-volume liquid handling in laboratory automation. 

Part 5 : Laboratory automation – solid dispensing. 

Part 6: Automated heating, shaking and mixing. 

Part 7: Automation of polymerase chain reaction (PCR).

Part 8 : Automated centrifugation. 

Part 9 : Automated filtration. 

Part 10: Laboratory automation – Automated sonication and evaporation.

Part 11: Laboratory automation – Automated incubation.

Contact

Prof. Dr.-Ing. habil. Kerstin Thurow
Center for Life Science Automation University of Rostock, Germany
www.celisca.de
[email protected]

References:

[1] Faraji, M., Yamini, Y., Gholami M. (2019). Recent Advances and Trends in Applications of Solid-Phase Extraction Techniques in Food and Environmental Analysis. Chromatographia. 82(50), 1207-1249. DOI: 10.1007/s10337-019-03726-9.

[2] Thurman, E. M., Mills, M.S. (1998). Solid-Phase Extraction – Principles and Practice. In: J. D. Winefordner (Hrsg.): Chemical Analysis, Volume 147, A Series of Monographs on Analytical Chemistry and its Applications. John Wiley & Sons, Inc., New York/Chichester/Weinheim/Brisbane/Singapur/Toronto, ISBN 0-471-61422-X.

[3] Schwedt, G. (1996). Festphasenextraktion als Probenvorbereitung für die Chromatographie. Nachrichten aus Chemie, Technik und Laboratorium. 44(4), M17-M22. DOI: 10.1002/nadc.19960440435.

[4] Fontanals, N., Marcé, R. M., Borulli, F. (2019). Materials for Solid-Phase Extraction of Organic Compounds. Separations. 6(4), 56. DOI: 10.3390/separations6040056.

[5] Thurow, K., Bach, A., Junginger, S. (2020). Parallele positive-pressure Festphasen-Extraktion - Eine Übersicht. Biospektrum, 26(5), 550-552. DOI: 10.1007/s12268-020-1441-z.

[6] Bach, A., Fleischer, H., Wijayawardena, B., Thurow, K. (2021). Optimization of Automated Sample Preparation for Vitamin D Determination on a Biomek i7 Workstation. SLAS Technologies. 26(6), 615-629. DOI: 10.1177/24726303211030291.

[7] Torres, A., Ravenelle, R.M. (2017). High-Throughput Filtration Using the MPE2 for Cannabinoid Analysis. Application Note. Hamilton (Reno, NV, USA).

[8] Thurow, K., Roddelkopf, T., Rohde, M., Bartel, J., Fleischer, H. (2020). Automatisierte Bestimmung von Dentalmaterialien aus Speichel. Biospektrum, 26 (2), 170-173. DOI: 10.1007/s12268-020-1347-9.

[9] Gallert, C., Vorberg, E., Roddelkopf, T., Junginger, S., Fleischer, H., Thurow, K. (2015). Evaluation of an Automated Solid-Phase Extraction Method Using Positive Pressure. American Pharmaceutical Review.

[10] Bach, A., Fleischer, H., Thurow, K. (2023). Comparison of Miniaturized Chromatographic Columns and 96-Well Plates for Automated Antibody Purification under Economic and Sustainable Aspects. Separations. 10(8), 447. DOI: 10.3390/separations10080447.

[11] Decker E. L., Reski R. (2008). Current achievements in the production of complex biopharmaceuticals with moss bioreactor. Bioprocess and Biosystems Engineering; 31(1), 3-9. DOI: 10.1007/s00449-007-0151-y.