Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying the length of the spacer arm (SA) linking the ligand to membrane surface. The membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi- component systems, in the batch mode. PAs are therapeutic agents for thrombovascular disorders. The membrane containing pABA linked through 7-atoms SA was found to bind up to 26-times higher amounts of PA per mg immobilized ligand as compared to membranes linked to pABA through 5- or 14- atoms long SA, from pure PA solution as well as the conditioned HeLa cell culture media. The significantly lower ligand density on the membrane surface in case of the 7-atoms SA might also have contributed to the higher PA binding due to the exclusion of crowding effect. Up to 69-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use.

Para-aminobenzamidine linked regenerated cellulose membranes for protein purification

245th National Meeting (2013)

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