Ultra-Fast Middle-Up Reversed Phase Liquid Chromatography Analysis of Complex Bispecific Antibodies Obtained in Less Than One Minute

Abstract

This work illustrates the benefits and limitations of using ultra-short reversed phase liquid chromatography (RPLC) columns for the characterization of various complex bispecific antibodies after prolonged thermal stress at the middle-up level of analysis. First, we have demonstrated that alternative organic modifiers, such as isopropanol, can be used in RPLC mode without generating excessive pressure, thanks to the prototype 10 × 2.1 mm, 2.7 µm particle column. However, compared to acetonitrile, the selectivity was not improved, at least for the selected biopharmaceutical products. Importantly, very fast separations (sub-1 min) of high quality were systematically obtained for the different samples when using a spectroscopic detector, but a severe loss of performance was observed with mass spectrometry (MS) detection due to dispersion effects. Based on these results, there is a clear need to improve the interfacing between LC and MS (shorter/thinner tubing) to mitigate band broadening.

Monitoring mAb proteoforms in mouse plasma using an automated immunocapture combined with top‐down and middle‐down mass spectrometry

“…Nonetheless, it should be noted that almost all the work published so far is based on the use of a large amount of starting material (a few micrograms), which is not sustainable for the analysis of in vivo biotransformation products. Improvements in stationary phases [49][50][51][52][53][54][55], low-flow chromatography, and alternative front-end separations [56][57][58] have recently opened the way to higher sensitivity studies. For instance, both the qualitative and quantitative top-down and middle-down analysis of biotherapeutics extracted from plasma has been described in the last years [59][60][61][62][63].…”