Evaluation of phenylboronate agarose for industrial-scale purification of erythropoietin from mammalian cell cultures.
The search for novel, cost-effective ways to produce erythropoietin (Epo), the world top-selling biopharmaceutical, is a major challenge for today's biotechnology industry. However, Epo's high glycosylation content (almost 40% of total mass) and the requirement for sialic acid for optimal in vivo activity still make mammalian cells the expression system of choice. In contrast to the abundance of reports on Epo production, robust, cost-effective methods for large-scale Epo purification can hardly be found in literature. To fill this gap, we describe here a process specifically studied for industrial-scale purification of the protein. Our method is based on the ability of phenylboronate agarose (PBA) to form reversible complexes with 1,2-cis-diol-containing molecules, like sugars in glycoproteins. Finding that additional factors (i.e., ionic and hydrophobic interactions) contribute to the Epo-PBA binding reaction, chromatography conditions have been optimized in scale-down experiments to improve selectivity and yield. As a result, the high performance of affinity chromatography has been achieved using a support possessing the robustness, chemical stability and low cost of a small synthetic ligand. By adding an anion exchange chromatography step and gel filtration for polishing, a pure and active product can easily be obtained by an integrated, start-to-end process optimized for industrial-scale operations.
Zanette
Dino
D
Lepetit Research Center, now BioSearch Italia, 21040 Gerenzano, Italy.
Soffientini
Adolfo
A
Sottani
Cristina
C
Sarubbi
Edoardo
E
D000818:Animals; D002478:Cells, Cultured; D002847:Chromatography, Agarose / Q000379:methods*; D004921:Erythropoietin / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D005240:Feasibility Studies; D008322:Mammals; D010865:Pilot Projects; D011786:Quality Control; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000031:analogs & derivatives*
D004921:Erythropoietin; C035597:phenylboronic acid-sepharose; D012685:Sepharose