ARTICLE IN PRESS JID: CHROMA [m5G;January 21, 2020;16:12] Journal of Chromatography A xxx (xxxx) xxx Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system Bastian Franke a,∗ , Tuomo Frigård b , Stephan Grzesiek a , Shin Isogai a,∗ a Focal Area Structural Biology and Biophysics, Biozentrum, University of Basel, 4056 Basel, Switzerland b GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 24 Uppsala, Sweden a r t i c l e i n f o Article history: Received 6 October 2019 Revised 12 December 2019 Accepted 31 December 2019 Keywords: ALIAS autosampler Automated protein purification In-line sample dilution Ion-exchange chromatography Green fluorescent protein G protein-coupled receptors a b s t r a c t Protein purification processes in basic research using ÄKTA TM liquid chromatography systems are often limited to single sample injections and simple one-column purifications. Because many target proteins in structural biology require complex purification protocols the work easily becomes laborious. To streamline and accelerate downstream protein production, an ALIAS TM autosampler and a modular sample in-line di- lution process coupled to ion-exchange chromatography were incorporated into the workflow to automate two of the most commonly performed purification strategies - ion-exchange to size exclusion and nickel- ion metal affinity to size exclusion. The chromatographic setup enabled purification of a large array of cytosolic and membrane proteins from small-scale expression cultures produced in insect cells necessary to develop and optimize isotope-labeling strategies for nuclear magnetic resonance spectroscopy applica- tions, resulting in a reduction in experiment time of about 20% per run for both cytosolic and membrane protein purification schemes. However, when queuing multiple samples the throughput increased by 66% and 75%, respectively. In addition, a novel system configuration is presented, where two column valves can be operated independently. This allows for the design of purification loops to increase purity of the target protein. © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. (http://creativecommons.org/licenses/by/4.0/) 1. Introduction ÄKTA TM chromatography systems have been designed to facili- tate protein purification processes, however laborious manual op- erations such as transfer, desalting and concentration of sample in between the purification runs are still quite common amongst research groups. The modular ÄKTAxpress instrument has simpli- fied repetitive high-throughput processes and automated multi- column purification protocols where routine production is needed [1], but the platform lacks flexibility and versatility. Several other studies describe more tailored multi-column purification schemes for specific target use [2], or generic protein purification machin- ery to aim for lean downstream development processes [3–6]. In all the cases, it involves highly modified ÄKTA instruments, addi- tional modules and convoluted flow paths that restrict rapid adap- tation of automation methods for other downstream purification experiments. Therefore, it is necessary to develop various use cases and building blocks further using standard hardware to; 1. facilite ∗ Corresponding authors. E-mail addresses: bastian.franke.pub@gmail.com (B. Franke), shin.isogai.pub@ gmail.com (S. Isogai). a day-to-day use of automation in downstream processes, and 2. provide a development scientist means to achieve flexible imple- mentation of automation in a limited amount of time. It is also crucial to establish closer collaborations between the academic and industrial research communities to facilitate flexible and adapted solutions for specific needs. In addition, adaptive system configu- rations and application dependent functionality is setting a new stretch goal for this to succeed. The introduction of the ÄKTA 3D Kit for the old discontinued ÄKTAexplorer range offered the first commercially available plat- form for facilitating various multistep protocols for the purifica- tion of up to 6 different His-or GST-tagged proteins on a day-to- day basis. However, the use of desalting (DS) columns and cap- illary loops not only restricted sample volume during purifica- tion but also lead to dilution of sample due to long flow paths during column-to-column transfer [6]. In a more recent study, a semi-preparative multistep purification method was performed on a commonly available ÄKTA pure chromatography system using a modular in-line dilution method coupled to ion-exchange (IEX) chromatography, which allowed for intermediate sample trans- fer as well as volumetric operations (dilution and concentration) [7]. This way, four G protein-coupled receptors (GPCRs) were https://doi.org/10.1016/j.chroma.2019.460846 0021-9673/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. (http://creativecommons.org/licenses/by/4.0/) Please cite this article as: B. Franke, T. Frigård and S. Grzesiek et al., Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460846