Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high-gradient magnetic separation.
Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed-bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed 'rotor-stator' type High Gradient Magnetic Fishing (HGMF) system for large-scale application, currently up to 100 g of magnetic adsorbents. Initially, the separation process design was optimized for binding and elution conditions for the target protein in mL scale. Subsequently, the magnetic filter for particle separation was characterized. Based on these results, a purification process for eCG was designed consisting of (i) pretreatment of the horse serum; (ii) binding of the target protein to magnetic ion exchange adsorbents in a batch reactor; (iii) recovery of loaded functionalized adsorbents from the pretreated solution using HGMF; (iv) washing of loaded adsorbents to remove unbound proteins; (v) elution of the target protein. Finally, the complete HGMF process was automated and conducted with either multiple single-cycles or multicycle operation of four sequential cycles, using batches of pretreated serum of up to 20 L. eCG purification with yields of approximately 53% from single HGMF cycles and up to 80% from multicycle experiments were reached, with purification and concentration factors of around 2,500 and 6.7, respectively.
Dept. of Chemical Engineering, Loughborough University, Loughborough, United Kingdom LE11 3TU.
Christine
C
Müller
Elena
E
Heidenreich
Matthias
M
Franzreb
Katrin
K
Frankenfeld
D000818:Animals; D001709:Biotechnology; D006063:Chorionic Gonadotropin / Q000097:blood* / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003959:Diamines / Q000737:chemistry; D005260:Female; D006736:Horses; D059346:Magnets / Q000737:chemistry*; D051379:Mice; D051381:Rats
D006063:Chorionic Gonadotropin; D003959:Diamines
equine chorionic gonadotropin; high gradient magnetic fishing; purification; serum