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

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 alternativ...

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Bibliographic Details
Published in:Pharmaceutics 2022-10, Vol.14 (11), p.2315
Main Authors: Murisier, Amarande, D’Atri, Valentina, Pirner, Sebastian, Larraillet, Vincent, Fekete, Szabolcs, Lauber, Matthew, Guillarme, Davy
Format: Article
Language:English
Subjects:
Antibodies
bispecific antibodies
Chromatography
Communication
improved selectivity
Ions
Liquid chromatography
method robustness
Molecular biology
Proteins
reversed-phase liquid chromatography
Software
ultra-short columns
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Summary: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.
ISSN:1999-4923
1999-4923
DOI:10.3390/pharmaceutics14112315