Proof-of-concept: Ultrafiltration as a pre-treatment for the anion exchange chromatography of RuBisCO from alfalfa

Alfalfa is gaining interest as an alternative protein source. European Food Safety Authority (EFSA) approval of alfalfa protein concentrate as novel food states presence of metabolites as a barrier for higher amount of consumption. A promising method for protein isolation is anion exchange chromatography, which is scalable through expanded bed chromatography. In this study, ultrafiltration-buffer exchange (UBE) and ultrafiltration-diafiltration was applied as an upstream and downstream processing to anion exchange chromatography, respectively. The performance of these techniques was compared to that of lab scale dilution and centrifugal spin filters. The result showed that UBE shortened the chromatography duration by 50% and resulted in a cleaner column. However, downstream desalination through ultrafiltration-diafiltration resulted in concentrate with lower protein content (60.41±0.84%), compared to that obtained from centrifugal spin filters (90.49±1.34%), suggesting the need to carry out extra diafiltration steps. Untargeted metabolomics revealed that high absorption at 214 nm in the anion exchange flowthrough fraction is attributed to di- and tripeptides also present in the ultrafiltration permeates. Polyphenols present in ultrafiltration permeate were absent from both flowthrough and 225 mM fraction, suggesting that they might bind strongly to the column. Various carboxylic acids were present in the ultrafiltration permeate, chromatography flowthrough, and 225 mM NaCl fraction. Contaminants eluting at 225 mM NaCl with high absorbance at 214 nm were not successfully identified although their occurrence was lower in UBE samples. These results suggest that UBE is promising as pre-treatment for purification of RuBisCO from alfalfa using anion exchange chromatography. • Ultrafiltration-buffer exchange reduced chromatography duration by 50% • Ultrafiltration-buffer exchange reduced yellow color retained by column • Polyphenols did not elute during the chromatography, suggesting strong resin binding • High absorbance at 214 nm in flowthrough is associated with peptides • Ultrafiltration-diafiltration resulted in lower purity compared to spin filters