Automated optimization of resin selection, wash parameters, and elution parameters for protein A-based purifications
Year: 2025
Type: article
Abstract: Automated protein purification is essential for meeting the growing demands of high-throughput sample processing capabilities, especially when screening multiple parameters. For instance, protein A-based affinity chromatography is an important downstream antibody purification process that can be automated to maintain high reproducibility with minimized human error. This purification step can clear undesirable host cell proteins (HCP) from Chinese hamster ovary (CHO) cells and capture target monoclonal antibodies, developed for a broad assortment of therapeutic applications, from antibody-expressing cell culture fluid. Selecting optimized parameters for protein A purification is crucial to ensuring productive performance in terms of optimal binding, prevention of undesirable effects from host cell protein, and product aggregate reduction. Herein, we demonstrate a faster and lower cost automated micro-purification workflow to accelerate the screening and selection of resin, wash, and elution parameters for protein A chromatography. In testing three monoclonal antibodies (mAb) and one bispecific monoclonal antibody (bsAb), we observed marked variability in purity and HCP clearance based on the wash buffer and elution pH. We also report that modified intermediate wash buffers containing sodium chloride minimized HCP contamination of eluted samples while preserving high monomer yields. Additionally, we identify differences in performance between resins, including the ability of Praesto Jetted A50 HipH to maintain higher yields at elution pH at which the yields of other resins declined (>pH 4.2). These results highlight the utility of automated microscale purification in reducing time and resource consumption involved in optimizing and executing downstream antibody bioprocessing. • A dispersive solid-phase extraction pipette tip on automated liquid handlers for rapid antibody purification process screenings • Robust micro-scale optimization reduces antibody purification process screening costs • Automated ProA resin workflows based on yield, HCP removal, and monomeric content • Salt and denaturant wash additives influence product purity and stability • Screening ProA resin properties can extend operable elution pH window and lower HCP contamination
Source: New Biotechnology
Authors: Gracie K. Anderson, Mark Hicks, Penny Hamlyn, L. Andrew Lee, Patrick A. Kates
Institution: Ecolab (United Kingdom)
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,670
OpenAlex ID: W4416275656