Modulation of physicochemical properties of magnetic agarose microspheres by hydrolysis-suppressive sequential crosslinking

Abstract

Separation and purification of target proteins are essential for drug discovery and development. A magnetic separation has been exploited due to its high efficiency. Despite many studies on chemical crosslinking of a magnetic agarose microsphere (MAM), the study on a crosslinking reaction preventing MAM from hydrolytic degradation during the reaction and the effects of the crosslinks on the physicochemical and electrochemical properties of the crosslinked MAM (CL-MAM) has rarely been reported. In our study, CL-MAM was prepared using a sequential crosslinking by which MAM was crosslinked using epichlorohydrin (ECH) in DMSO after crosslinking the MAM by 1,3-dichloro-2-propanol (DCP) for a short time. Such a crosslinking prevented the hydrolytic degradation of DCP-crosslinked MAM ( D -CL-MAM) and DCP and ECH-crosslinked MAM (DE-CL-MAM). MAM had a mean diameter of 110 ± 57.42 µm and its morphology was not affected by the crosslinking. In FT-IR analysis, increase of peak intensity at 1037 cm −1 confirmed newly formed C-O-C bonds in the agarose of CL-MAM. Thermal stability of MAM increased by both DCP crosslinking and subsequent ECH crosslinking of the agarose. While T g of MAM was not shifted by the crosslinking, the onset temperatures of glass transition and thermal decomposition increased by DCP crosslinking and further increased by DCP and ECH-crosslinking. Stiffness (S) and elastic modulus (E) of MAM were enhanced by DCP crosslinking and subsequent ECH crosslinking, indicating a half of ECH formed crosslinks between agarose mainchains while the other half crosslinked adjacent pyranose rings in the agarose mainchain. Permittivity analysis revealed that α relaxation temperature of the agarose film corresponded to T g determined by DSC analysis and the relaxation intensity decreased with increasing crosslink density of the agarose. The higher swelling ratio of DE-CL-MAM compared to D -CL-MAM was attributed to greater S and E values of DE-CL-MAM. The hydrolysis-repressive sequential crosslinking of MAM using DCP and ECH can be a feasible approach to modulate physicochemical and electrochemical properties of MAM. • Hydrolysis-suppressive crosslinkings by DCP and ECH were effective to crosslink MAM. • DCP-crosslinked MAM had enough stability to be crosslinkable by ECH in DMSO. • The sequential crosslinkings did not induce morphological changes of the MAM. • The crosslinking improved thermal stability of MAM, not influencing T g . • Mechanical and swelling properties of crosslinked MAM depended on degree of crosslinks.