Grafting hydrophobic and affinity interaction ligands on membrane adsorbers: A close-up “view” by X-ray photoelectron spectroscopy

The surface chemistry of chromatographic media plays a crucial role in their selectivity and binding capacities, enabling, or not, their use for the purification of therapeutic biomolecules. This work focuses on the XPS analysis of two different chromatographic membrane adsorbers to be integrated in the downstream processing of pDNA: (i) by hydrophobic interaction – alkyl and phenyl ligands, and (ii) by phenylboronate affinity interaction.The analysis of the Sartobind® epoxy precursor membrane evidenced both the major C 1s components expected for a cellulose membrane, at 286.7 and 288.1 eV as well as its major O 1s components at 532.9 and 533.5 eV. Also present in the C 1s region a peak at 289.0 ± 0.2 eV attributed to an ester group used as reinforcement of the cellulose and, likely derived from a carbonyldiimidazole (CDI) pre-activation. Traces of nitrogen (N/C ∼0.0035) helped corroborating the latter premise. The subsequent grafting to obtain a hydrophobic alkyl based membrane was followed by XPS. Global quantitative results attested the success of these reactions. Calculated atomic ratios for N/C and N/O bestowed information on the arrangement of the ligands during grafting and elucidaded the chromatographic behavior.The phenyl membrane adsorber presented the typical aromatic peak at 284.7 eV. On par with the previous Sartobind® epoxy membrane, the peak at ∼289.0 eV assigned to an ester group was present.Functionalization with 3-aminophenylboronic acid used two strategies one in which it was directly linked to the surface and an analogous strategy using sorbitol as shielding agent to prevent reaction of the boronate acid of the ligand with the epoxy. Quantitative XPS analysis demonstrated the fragility of this ligand, suffering from boronate hydrolysis, and the inefficacy of the sorbitol as shielding agent; concerning chromatography, the sorbitol methodology demonstrated a slight improvement.

► XPS of ligands grafting in membrane adsorbers for plasmid DNA purification.
► Sequential derivatization steps determine ligand relative position towards surface.
► XPS of a commercial phenyl membrane adsorber.
► XPS contribution of ester group in cellulose membrane adsorbers surface chemistry.
► Weakly bound affinity phenyl boronate moieties in prospective RNA adsorbers.