Effect of pore size on performance of monolithic tube chromatography of large biomolecules

Abstract

Effect of pore size on the performance of ion-exchange monolith tube chromatography of large biomolecules was investigated. Radial flow 1 mL polymer based monolith tubes of different pore sizes (1.5, 2, and 6 μm) were tested with model samples such as 20 mer poly T-DNA, basic proteins, and acidic proteins (molecular weight 14 000-670 000). Pressure drop, pH transient, the number of binding site, dynamic binding capacity, and peak width were examined. Pressure drop-flow rate curves and dynamic binding capacity values were well correlated with the nominal pore size. While duration of the pH transient curves depends on the pore size, it was found that pH duration normalized on estimated surface area was constant, indicating that the ligand density is the same. This was also confirmed by the constant number of binding site values being independent of pore size. The peak width values were similar to those for axial flow monolith chromatography. These results showed that it is easy to scale up axial flow monolith chromatography to radial flow monolith tube chromatography by choosing the right pore size in terms of the pressure drop and capacity.

Fast isolation of highly specific population of platelet-derived extracellular vesicles from blood plasma by affinity monolithic column, immobilized with anti-human CD61 antibody

“…A high affinity ligand attached to the monolith offers a useful system for fast and selective isolation of EVs. The monoliths with pore sizes of over 1 µm provide convective transport through the channels [9] with high flow rates [8], resulting in short separation times [10], and allow the isolation of large biomolecules without losing biological activity [11]. The monolithic columns, and especially poly (glycidyl methacrylate) ones, have usually unique separation properties for the large biomolecule separation [12][13][14][15], which makes them also suitable for the isolation of EVs.…”

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

“…DBCs around 7 mg pDNA per mL of column were consistent with expectations and literature data [11] for the smallest, 7.3 kbp large pAAV2/8 molecule. The DBCs of columns with flow channel diameters of 3 and 6 µm were expectedly lower in accordance with the reduction of the surface area (Table 2) of the monoliths with larger channels [29, 31].…”

“…Permeability increase was proportional to channel size and inversely proportional to surface area (see Figure S2). DBC for a standard protein biomolecule, such as BSA, was proportional to surface area and inversely proportional to channel size and according to the well-known literature data [29,30].…”