Sample transformation in online separations: how chemical conversion advances analytical technology

van der Zon, Annika A. M.; Verduin, Joshka; van den Hurk, Rick S.; Gargano, Andrea F. G.; Pirok, Bob W. J.

doi:10.1039/d3cc03599a

Cited by 2 publications

(2 citation statements)

References 171 publications

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“…Consequently, mD‐LC‐MS systems were developed to automate this workflow by splitting peaks online and automatically transferring them to the next separation dimension. This method offers major advantages over conventional offline analytical peak characterization, such as a significant reduction of the analysis time and the need for smaller sample quantities, while presenting fewer method‐induced artifacts [29].…”

Section: Md‐lc‐ms For Antibody Characterizationmentioning

confidence: 99%

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

Bouvarel,

Camperi,

Guillarme

2024

J of Separation Science

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This review provides an overview of the latest advancements and applications in multi‐dimensional liquid chromatography coupled with mass spectrometry (mD‐LC‐MS), covering aspects such as inter‐laboratory studies, digestion strategy, trapping column, and multi‐level analysis. The shift from an offline to an online workflow reduces sample processing artifacts, analytical variability, analysis time, and the labor required for data acquisition. Over the past few years, this technique has demonstrated sufficient maturity for application across a diverse range of complex products. Moreover, there is potential for this strategy to evolve into an integrated process analytical technology tool for the real‐time monitoring of monoclonal antibody quality. This review also identifies emerging trends, including its application to new modalities, the possibility of evaluating biological activity within the mD‐LC set‐up, and the consideration of multi‐dimensional capillary electrophoresis as an alternative to mD‐LC. As mD‐LC‐MS continues to evolve and integrate emerging trends, it holds the potential to shape the next generation of analytical tools, offering exciting possibilities for enhanced characterization and monitoring of complex biopharmaceutical products.

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Section: Md‐lc‐ms For Antibody Characterizationmentioning

confidence: 99%

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

Bouvarel,

Camperi,

Guillarme

2024

J of Separation Science

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“…Variation in the charge heterogeneity has been shown to alter the in vitro and in vivo properties of mAbs [5]. To characterize their proteoforms, mAbs are either analyzed via bottom-up (peptide level), middle-up (reduction, fragments), or intact protein approaches [6]. Charge variant analysis (CVA) is typically conducted at the intact proteoform level.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Monoclonal Antibody Charge Variants under Near-Native Separation Conditions using Nanoflow Sheath Liquid Capillary Electrophoresis-Mass Spectrometry

van der Zon,

Höchsmann,

Bos

et al. 2024

Preprint

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Monoclonal antibodies (mAbs) undergo multiple post-translational modifications (PTMs), e.g., charge variants, oxidation, etc., during production and storage, necessitating evaluation of the resulting PTMs as critical quality attributes (CQA) for protein quality and safety. Charge variants PTMs can be separated using capillary zone electrophoresis (CZE). The CZE EACA method developed by He et al. (2011) with UV detection is applied routinely in the pharmaceutical industry for analyzing charge variants. However, the method cannot be directly hyphenated with mass spectrometry (MS), preventing direct identification of separated charge variants due to the non-volatile background electrolyte (BGE), which hinders reliable charge variant identification. This study presents a CZE-UV/MS method using a neutral static capillary coating of hydroxypropyl methylcellulose combined with a volatile BGE at pH 5.0 to allow MS-compatible mAb charge variant separations. The effect of several parameters, including pH and concentration of BGE, voltage, and injected mAb concentrations in terms of separation performance on a panel of mAbs was investigated. The final method was tested with mAbs of IgG subclasses (IgG1 and IgG4) with different pI (7.4-9.2), and degrees of heterogeneity. Basic and acidic variants were separated from the parent mAb using a BGE of 50 mM acetic acid adjusted at pH 5.0. A linear correlation in relative charge variant abundance was obtained between our method and the EACA method. CZE-MS coupling was done using the nanoCEasy, a low-flow sheath liquid interface, and allowed the identification and quantitation of various low-abundance variants (<10% relative abundance with respect to the main compound). This method uses volatile buffers and operates at pH closer to non-denaturing conditions (pH 5.0), allowing for flexibility in hyphenation with MS. This method can be a useful tool for in-depth charge variants characterization of mAbs.

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Sample transformation in online separations: how chemical conversion advances analytical technology

Abstract: While the advent of modern analytical technology has allowed scientists to determine the complexity of mixtures, it also spurred the demand to understand these sophisticated mixtures better. Chemical transformation can...

Cited by 2 publications

References 171 publications

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

Characterization of Monoclonal Antibody Charge Variants under Near-Native Separation Conditions using Nanoflow Sheath Liquid Capillary Electrophoresis-Mass Spectrometry

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