Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high‐gradient magnetic separation

Abstract

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.

High-gradient magnetic separation for technical scale protein recovery using low cost magnetic nanoparticles

“…First results from a pilot plant similar to ours were published very recently and dealt with the multicycle purification of equine chorionic Gonadotropin (eCG) using microparticles [29]. Previous publications had already reported on studies carried out with a smaller rotor stator system: Brown and collaborators showed the suitability of HGMS for a technical application [30], whereas Müller and co-authors isolated eCG and compared the HGMS process with the technically established fixed-bed chromatography [31].…”

“…Our model system makes it possible to show the potential of HGMS as an useful bioprocessing alternative. Nevertheless, the usual systems have lower initial target protein concentration and would achieve better concentration and purification factors, as demonstrated by Müller et al for gonadotropin separation with microparticles [29].…”

“…A recent publication presenting first experiments with a parallel prototype by another scientific group included a detailed description of the separator [29], as already mentioned in the introduction. We just summarize here briefly main properties of the system.…”

First comprehensive view on a magnetic separation based protein purification processes: From process development to cleaning validation of a GMP‐ready magnetic separator

“…The device allows direct capturing and purification of MOI from crude feedstocks such as cultivation broth or natural sources for example, blood or blood serum [30,31]. An alternative purification process via magnetic separation has been presented by Müller et al, saving 2/3 of the solvent by using magnetic anionic exchange particles after a first precipitation procedure [37,38]. As an example, for the advantages of HGMS processes, the purification of the glycoprotein equine chorionic gonadotropin (eCG) from pregnant mare serum has been studied recently.…”

Magnetic Separation on a New Level: Characterization and Performance Prediction of a cGMP Compliant “Rotor‐Stator” High‐Gradient Magnetic Separator

“…Magnetic separation of biomolecules is mainly employed on a lab scale for cell sorting, DNA purification and analytical applications . Nevertheless, there are several studies dealing with the purification of commercially interesting biomolecules via magnetic separation on various scales . For this purpose, custom‐made prototype magnetic separators have been developed with a wide variety of approaches of shape, sizes, and magnetic background field but still following the classical fixed matrix design.…”