Nicolas Jean, Rebekah Eastwood, Francesco Rao, James Errey and Karthik Rajasekar. Evotec (UK) Ltd.
This article describes how scientists at Evotec, an R&D biotech company specializing in integrated drug discovery and development, have implemented automated size-exclusion chromatography (SEC) to increase throughput and consistency in protein purification workflows. The work highlights how the ALIAS autosampler can be used to streamline routine SEC runs, supporting efficient sample handling at scale. The study also highlights how Cytiva chromatography solutions were applied within this automated setup to enable reliable and reproducible purification.
Introduction
Engineered proteins have found many applications over the decades, including the development of novel therapeutics, improvements in enzyme efficiency, combating pest resistance, discovery of new research tools, and others. Recent advances in generative artificial intelligence (AI)-driven methodologies, such as protein large language models (pLLMs), have reduced the data requirement for de novo protein design. Consequently, the field has experienced a surge of new protein designs over the past three years. To validate and refine these AI-models and designs, large experimental datasets are essential, highlighting the increased need for high-throughput (HT) protein expression and purification workflows.
Building on our experience in protein production from Cytiva, Evotec‘s department of Protein Sciences established a HT pipeline for the expression and purification of proteins from multiple hosts, including E. coli, insect and mammalian systems. Click here to watch a webinar on protein purification with parallelized automated workflows. This article focuses on the automation of the final purification step, size exclusion chromatography (SEC), to produce homogenous protein samples for downstream assays. We describe how the use of the ALIAS autosampler enables automation of more than fifty SEC runs per day.
Instrumentation setup for high-throughput sizing column with fractionation
Small-volume, high-resolution analytical sizing columns are routinely used to determine the hydrodynamic radii and apparent molecular weights of macromolecules. Click here to read Cytiva web article. These columns are readily available with various base matrices (Superdex™, Superose™, and Sephacryl™) and pore sizes to enable separation of macromolecules of different size ranges.
For protein constructs that express well, standard UV detection provides sufficient sensitivity for data analysis.
Fig 1. A standard instrumentation setup for a high-throughput analytical sizing column with UV detection
Incorporating the ALIAS autosampler to a standard ÄKTA™ setup increases throughput and process efficiency (Figure 1). The complete setup can be housed within a 4°C cabinet or cold room to maintain sample stability throughout the workflow. The autosampler supports three injection modes: full loop, partial loop fill and µL pick-up. We use the µL pick-up for its accuracy, using a 250 µL loop to enable loading of 100 µL sample.
Hight throughput production workflow
A standard HT protein expression is carried out in a plate format at 4 mL scale (Figure 2). The harvest, lysis and affinity purification steps are all performed in plate-based formats and typically followed by a buffer exchange step. This workflow does not separate unfolded or aggregated protein from correctly folded homogenous protein which is essential for quantitative activity analysis of designed proteins.
To address this, elution fractions from the affinity step were transferred to vials for direct injection onto SEC column using ALIAS autosampler. The UNICORN™ software enables a straightforward queuing strategy for sequential sample injection through its scouting functionality, ensuring that all data are collected and analysed within the same platform.
Fig 2. Standard high-throughput production workflow using an ALIAS autosampler. HT=hight-throughput, SEC=size-exclusion chromatography, QC=quality control, SDS-PAGE = Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis, MS = Mass Spectrometry.
In a typical protein design project, hundreds to thousands of proteins are tested. Figure 3. shows the overlays of 48 production runs performed overnight, demonstrating the capacity and reproducibility of the automated workflow. This automated workflow enables over 400 high-quality protein samples to be purified per week.
The workflow can be readily adapted to include membrane proteins or antibodies. Furthermore, the strategy described here can be modified to support expression screening with affinity ([Click here to see Cytiva’s prepacked chromatography columns] or solubility screening of membrane proteins [Click here to read web article].
Fig 3. An example of high-throughput purification (top panel). Elution profile of molecular weight standards (bottom panel). Elution profile of 48 variants of Evotec-test protein. Shaded region shows the correct oligomeric status of the protein that is fractionated and pooled.
Summary and conclusion
Highly optimized, automated HT purification workflows are necessary to support the growing number of protein designs emerging from protein engineering and design projects. Integration of the ALIAS autosampler enhances the efficiency of the automated SEC purification. Producing high quality, non-aggregated proteins at scale is important for generating clean experimental datasets, which in turn enables the refinement and continued improvement of pLLMs.
More information:
- Using ALIAS autosampler for solubilization screen of membrane proteins
- Every peak matters
- Explore and find the perfect ÄKTA™ system for your research needs
- Configure your ÄKTA™ chromatography system
- Prepacked SEC columns
- ALIAS autosampler
Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an affiliate doing business as Cytiva. ÄKTA, ÄKTA pure, UNICORN, Superdex, Superose, and Sephacryl are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. Any other trademarks are the property of their respective owners.