Enhanced recovery yield by utilizing an improved purification method for recombinant human follicle-stimulating hormone expressed in CHO cells: Applying CaptureSelect™-FSH affinity matrix

Abstract

The CaptureSelect™ FSH affinity matrix is a novel affinity chromatography resin, developed based on camel-derived VHH fragments, capable of capturing and purifying intact rhFSH with high efficacy and purity in a single step. Since some proteins bind very tightly to the CaptureSelect™ FSH affinity matrix, conventional methods for desorption are ineffectual, and purification efficiency is reduced. Therefore, the factors affecting the desorption of proteins have been investigated. Protein desorption was greatly improved by introducing pauses into the elution step, and using an elution buffer concentration between 2 and 3 M MgCl2 also increased protein desorption. However, in some cases, the concentration-dependent chaotropic properties of MgCl2 caused protein inactivation. After applying each 20 mL of the protein mixture, the flow rate was paused, which improved binding capacity. Eventually, an efficient purification method is developed, in which the formulation buffer dramatically enhances yield. The overall yield and purity were 68.8 and 97%, respectively. The purified rhFSH was identified and characterized by several physicochemical and biological methods, such as gel electrophoresis, western blotting, high-performance liquid chromatography, and mass spectrometry. Biological activity was determined by In Vitro Maturation, and finally, the quaternary structures and thermal stability were assayed.