Abstract
Certain biomolecules have proven to be difficult to analyze by liquid chromatography (LC), especially under certain chromatographic conditions. The separation of proteins in aqueous mobile phases is one such example because there is the potential for both hydrophobic and ionic secondary interactions to occur with chromatographic hardware to the detriment of peak recovery, peak shape, and the overall sensitivity of the LC analysis. To decrease non-specific adsorption and undesired secondary interactions between column hardware and biomolecules, we have developed and applied a new hydrophilically modified hybrid surface (h-HST) for size exclusion chromatography (SEC) and anion exchange (AEX) separations of proteins and nucleic acids. This surface incorporates additional oxygen and carbon atoms onto an ethylene bridge hybrid siloxane polymer. As a result, it exhibits reduced electrostatic properties and hydrophilicity that facilitates challenging aqueous separations. Flow injection tests with a phosphate buffer showed superior protein recovery from an h-HST frit when compared to unmodified ethylene-bridged hybrid HST, titanium, stainless steel, and PEEK frits. When applied to SEC of rituximab, ramucirumab, and trastuzumab emtansine with a 50 mM ammonium acetate buffer, this new hydrophilic chromatographic hardware yielded improved monomer and aggregate recovery, higher plate numbers, and more symmetrical peaks. AEX columns also benefited from h-HST hardware. An acidic mAb (eculizumab) showed improved recovery, more stable retention, and a sharper peak when eluted from an h-HST versus SS column. Moreover, AEX separations of intact mRNA samples (Cas9 and EPO mRNA) were improved, where it was seen that h-HST column hardware provided higher sensitivity and more repeatable peak areas from injection to injection. As such, there is significant potential in the use of h-HST chromatographic hardware to facilitate more robust and more sensitive analyses for a multitude of challenging separations and analytes.
Highlights
It is well known that certain solutes tend to bind to chromatographic hardware surfaces or to the stationary phase through non-specific or non-desired interactions.[1−5] These interactions can often be attributed to metal hardware and electrostatic interactions occurring between charged analytes and the metal-oxide layer that is present at the surface of the hardware
Titanium column hardware has been reported to result in higher recovery for protein aggregates in size exclusion chromatography (SEC) and for some test proteins in cation exchange chromatography (CEX).[7]
We explored the use of column hardware with a vapor deposition hybrid silica surface and investigated its applicability to hydrophilic interaction and reversed phase liquid chromatography.[11]
Summary
It is well known that certain solutes tend to bind to chromatographic hardware surfaces or to the stationary phase through non-specific or non-desired interactions.[1−5] These interactions can often be attributed to metal (e.g., stainless steel) hardware and electrostatic interactions occurring between charged analytes and the metal-oxide layer that is present at the surface of the hardware (e.g., column frit, tubing, column inner wall, etc.). For systems containing hydrophobic polymers, like polyether ether ketone (PEEK) or polytetrafluoroethylene (PTFE, Teflon) components, large solutes with hydrophobic moieties (e.g., peptides, proteins, protein conjugates, or especially protein aggregates) may show recovery issues. These adsorptive interactions increase according to the number of functional groups within and the size of the analyte molecule. PEEK-lined reversed phase (RP) columns have been reported to provide superior recovery compared to conventional hardware for nucleotides, intact and subunit monoclonal antibodies (mAbs), and some peptides and better aggregate recovery in SEC.[6,8] A metal-free flow path
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