In biopharmaceutical analysis, undesired secondary analyte/surface interactions have hindered performance in HPLC. Analytes that contain electron rich functional groups, such as phosphate and carboxylate groups, are susceptible to absorb onto surfaces along the stainless-steel flow path causing reduced resolution and recovery. To overcome these challenges, hardware surfaces were modified with a chemically resistant hybrid organic/inorganic barrier and used in the construction of a bio-inert HPLC system. In this study, the bio-inert HPLC platform was evaluated by analysing oligonucleotides and monoclonal antibodies and compared to a legacy HPLC system for improved resolution and recovery.

To gauge the improvements in resolution and recovery that the bio-inert HPLC platform provides, established methods that were previously analysed on the legacy HPLC instrument were scaled and transferred to the new platform. For the analysis of monoclonal antibody standards, size exclusion chromatography was deployed for size variant analysis. The bio-inert HPLC was able to resolve an embedded peak in the monomer and another LMWS, which was not detected using the legacy HPLC. This improved resolution between the monomer and LMWS enables more precise quantitation of aggregate and fragment impurities that could affect product efficacy. For the analysis of oligonucleotides, a standard containing different lengths of oligodeoxythymidines and GEM91, a fully phosphorothioated oligonucleotide, were analysed and compared to a legacy HPLC system utilizing an ion pairing RPLC gradient. The bio-inert HPLC had an approximate 15% increase in recovery of the oligonucleotide standards.  For GEM91, the increase in recovery enabled better resolution of impurity peaks for improved quantitation when compared to the legacy HPLC instrument.

In conclusion, the newly designed bio-inert HPLC system with hybrid organic/inorganic surfaces demonstrated improved resolution and recovery for oligonucleotides and monoclonal antibodies compared to legacy systems. The improved HPLC platform can be broadly deployed to analyse new modalities and traditional biotherapeutics for routine analysis during manufacturing.