Comparison of Purification Strategies of Three Horseradish Peroxidase Isoenzymes Recombinantly Produced in Pichia pastoris

Horseradish peroxidase (HRP), a versatile heme-containing glycoprotein frequently used in medical diagnostics, is primarily isolated from plant. This process involves filtration, salt precipitation and several chromatography steps, which is expensive, renders low yields and gives isoenzyme mixtures. Although single isoenzymes can be recombinantly produced in Pichia pastoris, they get hyper-glycosylated in the yeast rendering the downstream process cumbersome and thus not competitive. In this study, we analyzed the purification of three HRP isoenzymes differing in the number of N-glycosylation sites recombinantly produced in P. pastoris. We wanted to 1) determine potential correlations between the mode of protein production and the resulting product quality and downstream process, 2) investigate correlations between the number of N-glycosylation sites of HRP and the mode of purification, and 3) find the optimal purification strategy for the recombinantly produced HRP isoenzymes. Thus, we applied different cultivation strategies and tested downstream processes employing both particlebased resins as well as monolithic supports. We showed that the mode of cultivation affected product purity but not the subsequent downstream process. Purification of each of the three HRP isoenzymes using monolithic supports was successful and independent of the number of N-glycosylation sites, whereas purification by particle-based resins was highly affected by glycosylation. Summarizing, we demonstrated a novel feasibility of using monoliths operated in flow through mode for the purification of comparatively small biomolecules.