Magnetic Precipitation: A New Platform for Protein Purification

Abstract

One of the trends in downstream processing comprises the use of “anything‐but‐chromatography” methods to overcome the current downfalls of standard packed‐bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation is developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit are tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions are obtained when using PEG3350 as precipitant at 4 °C for 1 h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti‐TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies.

Direct capture and selective elution of a secreted polyglutamate‐tagged nanobody using bare magnetic nanoparticles

“…who combined protein precipitation through PEG with affinity interactions of antibodies and triazine‐functionalized magnetic particles. [ 28 ] We chose 10% PEG 6000 since smaller proteins tend to precipitate with larger PEG sizes and higher concentrations, and we found 10% PEG 6000 to be sufficient. [ 35,36 ] We investigated the influence of the PEG on the magnetic nanoparticles and on the proteins to further evaluate the observed phenomenon.…”

“…[ 27 ] Recently, researchers observed improved capturing efficiency with magnetic particles by combining it with precipitation, naming that approach ‘affinity magnetic precipitation’. [ 28 ] Santos et al. could enhance the yield and purity of captured antibodies by adding polyethylene glycol (PEG).…”

“…could enhance the yield and purity of captured antibodies by adding polyethylene glycol (PEG). [ 28 ] The approach by Santos et al. is based on functionalized magnetic particles, whereas the magnetic nanoparticles in this study are non‐functionalized.…”

“…is based on functionalized magnetic particles, whereas the magnetic nanoparticles in this study are non‐functionalized. [ 28 ] The combination of PEG and magnetic particles is also known in magnetic aqueous two‐phase systems (ATPS), where magnetic particles are known to enhance the phase separation [ 29,30 ] or bind proteins when being functionalized. [ 31–33 ] Aside those two downstream applications, PEG is commonly known to precipitate proteins by steric exclusion and even allows for an improved retention of proteins to the chromatographic material.…”

“…[27] Recently, researchers observed improved capturing efficiency with magnetic particles by combining it with precipitation, naming that approach 'affinity magnetic precipitation' . [28] Santos et al could enhance the yield and purity of captured antibodies by adding polyethylene glycol (PEG). [28] The approach by Santos et al is based on functionalized magnetic particles, whereas the magnetic nanoparticles in this study are non-functionalized.…”