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Evaluation of additives on reversed-phase chromatography of monoclonal antibodies using a 1000 Å stationary phase

McCalley, David V.; Guillarme, Davy


Davy Guillarme


© 2019 Elsevier B.V. A wide pore (1000 Å) diphenyl stationary phase was evaluated for the analysis of monoclonal antibodies (mAbs), comparing a conventional mobile phase of acetonitrile-water containing overall 0.1% trifluoracetic acid (TFA) with a similar mobile phase incorporating in addition 5% butanol. Alternatively, TFA was replaced by ammonium formate (AF) buffer (pH 3.0) and by methane sulfonic acid. Addition of 5% butanol to the mobile phase reduces the minimum temperature at which suitable UV analysis of the mAbs can be obtained from about 70 °C with TFA alone to about 60 °C thus potentially improving column lifetime and reducing the possibility of sample degradation. AF buffers produce satisfactory UV sensitivity at 70 °C and have the advantage of reducing signal suppression in mass spectrometry (MS). Some peak tailing was noted in comparison with TFA separations. Methane sulfonic acid at the same molar concentration as TFA produced the best chromatographic peaks, maintaining reasonable UV sensitivity down to 50 °C, also giving acceptable results even at only 3 mM concentration of the additive. The good results with this additive were attributed to its stronger acidity and consequent suppression of the ionisation of column silanols. Surprisingly, peak response (as measured by the size of the peaks) was rather poorly correlated with the peak capacity of the gradient analysis. A possible explanation is self-deactivation of active column sites by a portion of the sample.

Journal Article Type Article
Publication Date Jan 11, 2020
Journal Journal of Chromatography A
Print ISSN 0021-9673
Electronic ISSN 1873-3778
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 1610
Article Number 460562
APA6 Citation McCalley, D. V., & Guillarme, D. (2020). Evaluation of additives on reversed-phase chromatography of monoclonal antibodies using a 1000 Å stationary phase. Journal of Chromatography A, 1610,
Keywords Organic Chemistry; Analytical Chemistry; Biochemistry; General Medicine
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