New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies

Fekete, Szabolcs; Murisier, Amarande; Beck, Alain; Lawhorn, Jason; Ritchie, Harry; Boyes, Barry E.; Guillarme, Davy

doi:10.1016/j.chroma.2021.462050

Cited by 9 publications

(3 citation statements)

References 30 publications

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“…In the RPLC of proteins, acetonitrile remains the most widely used mobile phase component. However, as already demonstrated elsewhere [9], the chromatographic selectivity can possibly be altered by changing the nature of the organic modifier. This behavior could be attributed either to the modification of weak chemical interaction (acetonitrile is aprotic, while alcohol-based modifiers are protic, so H-bond interaction may be strongly modified) or to the fact that mobile phase viscosity could vary significantly when changing the nature of the organic solvent.…”

Section: Evaluation Of Alternative Mobile Phase Eluentsmentioning

confidence: 76%

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

et al. 2022

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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.

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Section: Evaluation Of Alternative Mobile Phase Eluentsmentioning

confidence: 76%

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

et al. 2022

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“…This stationary phase is highly bonded, and the ligands are very rigid, limiting secondary interactions and conformational heterogeneity of protein adsorption . Its qualities were demonstrated in LC-MS analyses of antibody biopharmaceuticals that involved a low or even zero concentration of TFA in the mobile phase and a mild column temperature. , Despite the extraordinary qualities of particle chemistries developed to respond to the demands of the pharmaceutical industry for columns for quality control of modern protein drugs, ,, poly(styrene- co -divinylbenzene) macroporous polymeric particles, which were introduced more than three decades ago, are still customary for protein separations in top-down proteomic studies. , We anticipate that top-down proteomics will largely benefit from stationary phase developments driven by the need for quality control of protein biopharmaceuticals.…”

Section: Rplc Methods For Bottom-up Proteomic Analysesmentioning

confidence: 99%

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial

et al. 2022

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The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

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“…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].…”

Section: Significance Statementmentioning

confidence: 99%

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

et al. 2023

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Monoclonal antibodies (mAbs) have established themselves as the leading biopharmaceutical therapeutic modality. Once the developability of a mAb drug candidate has been assessed, an important step is to check its in vivo stability through pharmacokinetics (PK) studies. The gold standard is ligand‐binding assay (LBA) and liquid chromatography‐mass spectrometry (LC‐MS) performed at the peptide level (bottom‐up approach). However, these analytical techniques do not allow to address the different mAb proteoforms that can arise from biotransformation. In recent years, top‐down and middle‐down mass spectrometry approaches have gained popularity to characterize proteins at the proteoform level but are not yet widely used for PK studies. We propose here a workflow based on an automated immunocapture followed by top‐down and middle‐down liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) approaches to characterize mAb proteoforms spiked in mouse plasma. We demonstrate the applicability of our workflow on a large concentration range using pembrolizumab as a model. We also compare the performance of two state‐of‐the‐art Orbitrap platforms (Tribrid Eclipse and Exploris 480) for these studies. The added value of our workflow for an accurate and sensitive characterization of mAb proteoforms in mouse plasma is highlighted.

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New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies

Cited by 9 publications

References 30 publications

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

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

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial

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

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