New stationary phases for high‐performance liquid chromatography of biomolecules

This work presents a study on the preparation and application of polymer monoliths for the liquid chromatography of biomolecules with a focus on the ion‐exchange (IEX) mode. As one important application of polymer monoliths in bioanalysis, charge heterogeneity profiling of monoclonal antibodies (mAbs) in different biopharmaceuticals was performed by developing an elution approach based on shallow pH gradient, generated using single component buffer systems as eluents through cation‐exchange (CEX) monoliths as stationary phases. A useful selection of small molecule buffer species is described that can be used within very narrow pH ranges (typically 1 pH unit) defined by their buffering capacity for producing controlled and smooth pH profiles when used together with porous polymer monoliths. The results obtained appeared to be consistent with those obtained by imaged capillary isoelectric focusing (iCE) in terms of both resolution and separation profile. The retention mechanism based on the trends observed for proteins at pH values higher than the electrophoretic pI, as well as the high resolution gains, were discussed using applicable theories. Very low ionic strength eluents also enabled direct coupling of the ion‐exchange chromatography (IEC) to mass spectrometer for further characterisations of mAbs. Although there are few reports of IEC‐MS technique for small proteins in which the IEX column is directly interfaced to the mass spectrometer, the employment of a linear pH gradient elution scheme directly interfaced to mass spectrometer for the analysis of large proteins such as mAbs is also unique in the present work. New polymer monoliths were prepared in 100 μm i.d. capillaries by thermally‐initiated co‐polymerisation of glycidyl methacrylate as reacting monomer and pentaerythritol triacrylate as a hydrophilic crosslinker. The monolith recipe and polymerisation conditions were optimised to obtain a homogeneous monolith with good mechanical stability and characteristics suitable for separation of biomacromolecules. Nevertheless, shrinkage of the material prevented making monoliths in a column with conventional dimensions. Post‐polymerisation modification of the monolith was performed via optimised reaction conditions in order to incorporate weak cation‐exchange (WCX) or strong cation‐exchange (SCX) functionalities using amine reagents respectively containing phosphoric acid or sulforic acid groups. Dynamic binding capacities up to 15.1 mg/mL were measured using lysozyme as a standard probe, which is comparable or greater from some of the commercially available columns. Compared to monoliths reported previously for the same purpose, the developed monoliths also demonstrated negligible hydrophobicity with separation efficiency of approximately 55,000 plates/m in isocratic separation of sample proteins. A versatile epoxy‐based monolith was synthesised in 100 μm i.d. capillaries by polycondensation polymerisation of glycidyl ether 100 with ethylenediamine using a porogenic system consisting of polyethylene glycol, MW = 1000, and 1‐decanol. Polymerisation was performed at 80 °C for 22 h. The resultant monolith possessed hydrophilic properties originating from the incorporation of hetero‐atoms in the monolith skeleton which was further strengthened by simple acid hydrolysis of residual epoxides, resulting in a mixed diol‐amino chemistry. The modified column was used successfully for hydrophilic interaction liquid chromatography (HILIC) of small molecule probes, such as nucleic acid bases and nucleosides, benzoic acid derivatives, as well as for peptides released from a tryptic digest of cytochrome c. The mixed mode chemistry allowed both hydrophilic partitioning and IEX interactions to contribute to the separation, providing flexibility in selectivity control. Residual epoxide groups were also exploited for incorporating a mixed IEX chemistry. Alternatively, the surface chemistry of the monolith pore surface rendered hydrophobic via grafting of a co‐polymerised hydrophobic hydrogel. The inherent hydrophilicity of the monolith scaffold also enabled high performance separation of proteins under IEX and hydrophobic interaction (HIC) modes and in the absence of nonspecific interactions