Current and future trends in reversed-phase liquid chromatography-mass spectrometry of therapeutic proteins

D’Atri, Valentina; Murisier, Amarande; Fekete, Szabolcs; Veuthey, Jean‐Luc; Guillarme, Davy

doi:10.1016/j.trac.2020.115962

Cited by 31 publications

(20 citation statements)

References 47 publications

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“… 22 − 24 Denaturing reversed-phase liquid chromatography (RPLC) is among the most widely used separation techniques with protein retention based on hydrophobic interactions. 25 Capillary zone electrophoresis (CZE) coupled to MS, in comparison, separates molecules based on charge and size characteristics within a capillary using an applied electric field in the presence of a background electrolyte. 26 Ion exchange is another charge-based online separation method that has recently been developed to study intact therapeutic proteins such as antibodies.…”

Section: Introductionmentioning

confidence: 99%

“…To better resolve heterogeneous samples (e.g., glycoproteins), liquid chromatography is often coupled to MS to separate proteins based on their chemical properties before MS detection. − Denaturing reversed-phase liquid chromatography (RPLC) is among the most widely used separation techniques with protein retention based on hydrophobic interactions . Capillary zone electrophoresis (CZE) coupled to MS, in comparison, separates molecules based on charge and size characteristics within a capillary using an applied electric field in the presence of a background electrolyte .…”

Section: Introductionmentioning

confidence: 99%

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Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain

et al. 2022

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SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein’s structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain

et al. 2022

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“…It is the preferred mode of chromatography for determining the relative hydrophobicity of mAbs and their variants and can monitor various types of PTMs of mAbs, such as degradation, fragmentation, misfolding, oxidation, carboxy-terminal heterogeneity, aspartic acid isomerization, and unpaired cysteines or free thiols . Although RPC is also used to analyze hydrophobic variants, most intact proteins tend to denature in the presence of high organic content as the organic solvents tend to disrupt the hydrophobic bonds present in proteins, thereby causing the native state to denature. , Further, undesired adsorption of intact proteins on the RPC stationary phase due to secondary electronic interactions and harsh operating conditions also leads to protein denaturation . Hence, HIC is preferred over RPC for analysis of hydrophobic variants in their native structure.…”

Section: Introductionmentioning

confidence: 99%

Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS

2022

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Charged heterogeneity of monoclonal antibody (mAb) products is regarded as a critical quality attribute (CQA) depending on its impact on the safety and efficacy profile of the product. Hence, manufacturers are expected to perform a comprehensive characterization of the charge heterogeneity to ensure that the manufactured product meets its specifications. Further, monitoring is also expected during the product lifecycle to demonstrate consistency in product quality. However, conventional analytical methods for characterization of hydrophobic and charge variants are nonvolatile salt-based and require manual fraction collection and desalting steps before analysis through mass spectrometry can be performed. In the present study, a workflow of a two-dimensional liquid chromatography method using mass spectrometry (MS)-compatible buffers coupled with native mass spectrometry was performed to characterize hydrophobic variants in the first dimension and charge variants in the second dimension without any need for manual fractionation. This novel two-dimensional (2D) hydrophobic interaction chromatography (HIC)-weak cation-exchange chromatography (WCX)-MS workflow identified 10 variants in mAb A, out of which 2 variants are exclusive to the 2D orthogonal method. Similarly, for mAb B, a total of 11 variants are identified, including 5 variants exclusive to the 2D orthogonal workflow. When compared to stand-alone, HIC resolved only 4 variants for both mAbs and WCX resolved 7 variants for mAb A and 6 variants for mAb B. In addition, the proposed method allows direct characterization of hydrophobic/charge variant peaks through native mass spectrometry in a single-run workflow.

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“…[22][23][24] Denaturing reversed-phase liquid chromatography (RPLC) is among the most widely used separation techniques with protein retention based on hydrophobic interactions. 25 Capillary zone electrophoresis (CZE) coupled to MS, in comparison, separates based on charge and size characteristics within a capillary using an applied electric field in the presence of a background electrolyte (BGE). 26 Ion exchange is another charge-based online separation method that has recently been developed to study intact therapeutic proteins such as antibodies.…”

Section: Introductionmentioning

confidence: 99%

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

Wilson

Bilbao

Wang

et al. 2022

Preprint

View full text Add to dashboard Cite

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein’s structure and function, and thus comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid-chromatography coupled to mass spectrometry (LCMS) has been widely used to characterize post-translational modifications in proteins including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogenous glycans at the intact protein level. Herein, we evaluated several online separation techniques: 1. C2 reverse-phase liquid chromatography (RPLC), 2. capillary zone electrophoresis (CZE), and 3. acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down MS. Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation, N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogenous glycoproteins for facile comparison of biosimilars in quality control applications.

show abstract

Current and future trends in reversed-phase liquid chromatography-mass spectrometry of therapeutic proteins

Cited by 31 publications

References 47 publications

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain

Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

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