Learn how Athebio AG used Cytiva™ Protein Select™ resin to purify recombinant proteins
We asked Athebio AG for their feedback on using our Cytiva™ Protein Select™ resin and self-cleaving tag, designed to simplify tagged protein purification and tag removal (Fig 1). Athebio AG is a biopharma company that enables next-generation targeting of biotherapeutics.
Fig 1. Traceless tag cleavage and tag removal in one step with Cytiva™ Protein Select™ chromatography resin completes your affinity purification.
Athebio's experience with Cytiva™ Protein Select™ resin
We asked for their feedback using our Cytiva™ Protein Select™ technology. Athebio AG is a biopharma company that enables next-generation targeting of biotherapeutics.
Tell us about yourself.
I am Simon Flückiger, Research Associate working at Athebio AG in Zürich, Switzerland. I have a background in molecular biology and bioprocess engineering, and I am passionate about finding simple, elegant solutions for complex problems.
Fig 2. Simon Flückiger, Athebio AG.
Tell us about your work.
I work in the following areas: industrial research, drug discovery, and process development.
We empower drug developers with our Athebody® DARPin technology platform, enabling novel biotherapeutic approaches across a variety of applications. In my role, I focus on delivering DARPin candidates for drug development to our partners. In yet another part of my role I am continuously striving to improve and optimize internal processes.
What sparked your interest in Cytiva™ Protein Select™ technology? What problem(s) were you hoping to solve?
Some of our activities at Athebio require tag-free protein purification. Using traditional methods, we were facing problems of tag-removal. I therefore searched for an elegant alternative to our current approach. Enabling a one-step purification with tag removal, the Cytiva™ Protein Select™ technology offered the opportunity to save time, material, and to develop a more reproducible process.
What has Cytiva™ Protein Select™ resin helped you to improve in your work?
The Cytiva™ Protein Select™ resin enabled me to develop a more reliable process. Using Cytiva™ Protein Select™ we save time and reduce the loss of material during the process. So far, we observed a high reproducibility of the process when switching between different proteins.
With which expression system(s) have you been using Cytiva™ Protein Select™ technology?
E. coli
What protein type(s) and size(s) did you purify with this technology?
We work with Athebody® DARPins (Designed Ankyrin Repeat Proteins), with sizes ranging from approx. 15 to 22 kDa.
What will you use your purified protein(s) for?
The proteins will be used in various assays to evaluate their therapeutic potential.
In one sentence, what was the best thing about Cytiva™ Protein Select™ resin?
I like the simplicity of the one-step purification process that provides you with pure, tag-free proteins, already in the right buffer.
How does using Cytiva™ Protein Select™ resin compare with your past way of working?
Our previous approach involved traditional affinity Immobilized Metal Affinity Chromatography (IMAC) purification combined with protease-based tag removal approaches. Using the Cytiva™ Protein Select™ resin, we were able to reduce hands-on time thanks to the one-step approach, reducing the time requirement from days to hours of work.
Please describe in further detail some key outcomes from your work with Cytiva™ Protein Select™ resin.
Before the shift to Cytiva™ Protein Select™, the purification consisted of an IMAC step, followed by protease cleavage of the His-tag, accompanied by multiple steps of buffer exchange and sample concentration. The purification with IMAC and tag-removal took three days of high hands-on time. Due to protein-to-protein variations, the whole process was very inconsistent and difficult to predict due to high losses of protein during tag removal and cleanup.
Using the Cytiva™ Protein Select™ technology, we purify up to 5 tag-free DARPins in parallel, in two days with 4 hours hands on time. The bacterial cells are first lysed, and cell debris is cleared by centrifugation. DARPins being thermostable, the cleared lysate is heat-treated to remove most of the host cell proteins. After another clearance step, the sample is loaded on the column. The purification procedure consists of four steps: Sample load, Column Wash, an on-column incubation overnight and elution (see Fig 3A). In the first trials the elution was contaminated with a protein running at approx. 70 kDa (highlighted red in Fig 3B), mass spectrometry analysis (finger printing) revealed that it was the Chaperonin Hsp70 from E. coli (dnaK). To release the chaperones from the DARPins, ATP and MgCl2 were added to the cleared lysate after heat treatment. This allowed to obtain an elution with pure protein as verified by SDS-PAGE (see Fig 3B), analytical SEC (Fig 3C) and MS (data not shown). The hazy appearance below the purified protein band, can likely be attributed to the high stability of the DARPins.
DARPins produced with the Cytiva™ Protein Select™ method performed equally or better than our original approach in any conditions we tried so far.
Fig 3. Athebio AG's purification and tag-removal of Cytiva™ Protein Select™ resin.
Is there anything else you'd like to add?
Thanks a lot for the great support that I received from Cytiva, during the implementation and feasibility study of the Cytiva™ Protein Select™ resin.
Your success is our success
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