44 February / March 2021
acid), significant tailing is observed for the T37 peak, obscuring the presence of small trailing peaks (see Figure 6B). In contrast, using both a system and column that incorporate MaxPeak HPS technology, the T37 peak is sharp and symmetric, allowing accurate quantification of the trailing peaks. These minor peaks arise from deamidated variants, and their areas relative to the main T37 peak are monitored for quality control of therapeutic monoclonal antibodies [25].
In an attempt to reduce the tailing of the T37 peptide peak when using conventional metal surface technology, the UPLC system was conditioned by washing with a 30% phosphoric acid solution for 20 min followed by flushing with water until the pH was neutral. This was found to temporarily reduce the tailing of the T37 peak, but the tailing factor increased over 48 h of exposure to the 0.1% formic acid mobile phases, indicating an increase in surface adsorption activity. This is shown in Figure 7. In contrast, with MaxPeak HPS technology similar results were obtained with and without the phosphoric acid wash, with consistent assay performance observed from beginning to end when this separation was performed over the same time period. Equally important is that the tailing factor was lower when using both a system and column that incorporate MaxPeak HPS technology, resulting in excellent resolution of the deamidation variants, allowing them to be accurately and reproducibly quantified.
Conclusions
These results demonstrate the challenges posed by interactions of analytes with the metal surfaces in conventional UPLC systems and columns. Mitigation of these interactions using time-consuming conditioning approaches can add hours to the analysis time, and don’t guarantee that accurate and reproducible results will be obtained. Using the new capabilities afforded by systems and columns that
incorporate MaxPeak HPS technology, separations of metal-sensitive analytes may now be carried out without extensive conditioning protocols, providing accurate and reproducible results in a much shorter time. As demonstrated here, this technology is important for a range of analyte classes, including acidic polar metabolites, oligonucleotides and acidic peptides. Other analytes containing phosphate and/ or carboxylate groups show similar benefits [16], as do some compounds containing additional electron-rich functional groups. Consequently, this technology is likely to be beneficial for a range of applications.
Acknowledgments
The authors thanks Mathew DeLano and Andrew Bates-Harrison for providing the MaxPeak HPS hardware and Gary Izzo and Glen McGrath for packing the columns used in these experiments.
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