Preparation and characterization of micro-cell membrane chromatographic column with N-hydroxysuccinimide group-modified silica-based porous layer open tubular capillary

Cell membrane chromatography (CMC) is an effective tool in screening active compounds from natural products and studying membrane protein interactions. Nevertheless, it always consumes a large amount of cells (e.g. 107-108) for column preparation. To overcome this, micro-CMC (mCMC), that employs a silica capillary as membrane carrier, was developed. However, both CMC and mCMC suffer from short column life span (e.g. 3 days), mainly due to the falling-off of cellular membranes (CMs). This has greatly limited further application of CMC and mCMC, especially when the cells are hard to obtain. To solve this, N-hydroxysuccinimide (NHS)-modified silica-based porous layer open tubular capillary was first prepared for mCMC. The NHS groups can easily react with amino groups on CMs to form a stable covalent bond under a mild condition. So, CMs immobilized on the NHS-modified capillary are less likely to fall off. To verify this, SKBR3/mCMC (Her2 positive) and BALL1/mCMC (CD20 positive) columns were prepared. Two monoclonal antibody drugs, trastuzumab (anti-Her2) and rituximab (anti-CD20), were selected as analytes to characterize the columns. As a result, NHS-modified column for mCMC can afford higher chromatographic retention than non-modified column. Besides, the column life span was significantly improved to more than 16 days for SKBR3/mCMC and 14 days for BALL1/mCMC, while the compared column showed a sharp decline in retention factor in first 3 days.