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New stationary phases for high‐ performance liquid chromatography of biomolecules


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Talebi, M 2013 , 'New stationary phases for high‐ performance liquid chromatography of biomolecules', PhD thesis, University of Tasmania.

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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.

Item Type: Thesis - PhD
Authors/Creators:Talebi, M
Keywords: Polymer monoliths, Epoxy-based monoliths, capillary monolithic columns,Liquid Chromatography-Mass spectrometry (LC-MS), Hydrophilic Interaction Liquid Chromatoghraphy (HILIC), pH-gradient ion-exchange chromatography, proteins, peptides
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