Low-gradient magnetic separation in biotechnology: automated high-flow affinity purification from crude E. coli lysates

Scaling up magnetic affinity purification from small-scale bead handling to automated preparative processing requires integrated control of particle retention and liquid exchange. A fully automated low-gradient magnetic separation (LGMS) platform was developed for protein purification from Escherichia coli lysates using micrometer-sized magnetic beads functionalized with Strep-Tactin (STMBs). Movable neodymium permanent magnets enable bead retention and resuspension without the need for a magnetizable high-gradient capture matrix. The workflow integrates capacity-based loading, gravimetric feed dosing, affinity capture, washing steps, competitive elution using biotin, and alkaline bead regeneration. Near-infrared (NIR) and ultraviolet (UV) inline sensors monitor particle clearance and protein-related process signals. Lab-scale experiments confirmed protein capture from unclarified lysate as well as the repeated reuse of beads following alkaline regeneration. An anti-hCD45 nanobody (NB) and Green fluorescent protein (GFP) were investigated in separate experimental sequences, each consisting of three consecutive cycles. In each case, 500 mL of centrifugally clarified, unfiltered lysate was processed with 22 mL of sedimented STMBs at a recirculation flow rate of approximately 1 L min −1 . Each sequence processed 1.5 L within approximately 4.5 h. Overall recoveries, relative to an affinity-chromatographic reference, were 91.5% for the NB and 92.9% for GFP. Reusing the same biotin elution pool increased final concentrations to 1.72 and 3.07 g L −1 , respectively, corresponding to approximately 2.8-fold enrichment compared to the feeds. Final size-exclusion chromatographic purities were 95.6% and 88.6%, respectively. These results provide a liter-scale proof of concept for automated magnetic affinity purification with integrated product concentration and provide the basis for further scale-up of LGMS-based downstream processing.