Ion-pair reversed-phase high performance liquid chromatography method for the quantification of isoaspartic acid in a monoclonal antibody

Kern, Wolfram; Mende, Robin; Denefeld, Blandine; Sackewitz, Mirko; Chelius, Dirk

doi:10.1016/j.jchromb.2014.02.017

Cited by 3 publications

(1 citation statement)

References 34 publications

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“…The last considered PTM is aspartic acid isomerization; this modification is particularly difficult to characterize. Indeed, the change of conformation of aspartic acid (Asp) could not induce a significant variation of affinity toward the reverse stationary phase and requires particular analytical methodologies giving access only to a specified aspect of the protein. − Furthermore, the conformation change does not induce a change in the mass of the peptide; thus, ESI-MS using hybrid analyzers such as a quadrupole-time-of-flight (Q-TOF) does not allow a determination of potential Asp isomerization. From the CESI-MS/MS data, extraction of the m / z ratio corresponding to a peptide potentially presenting Asp isomerization systematically exhibited two consecutive peaks as shown in Figure .…”

Section: Resultsmentioning

confidence: 99%

Full Antibody Primary Structure and Microvariant Characterization in a Single Injection Using Transient Isotachophoresis and Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry

Gahoual

Busnel²,

Beck

et al. 2014

Anal. Chem.

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Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS.

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Section: Resultsmentioning

confidence: 99%

Full Antibody Primary Structure and Microvariant Characterization in a Single Injection Using Transient Isotachophoresis and Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry

Gahoual

Busnel²,

Beck

et al. 2014

Anal. Chem.

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Technological Platforms to Study Plant Lipidomics

Afzal

Naz

Ayub

et al. 2016

Plant Omics: Trends and Applications

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Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy

et al. 2021

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The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pKa values of isoAsp and C-terminal Asp in short peptides. The characteristic 1H-13C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4–5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.

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Ion-pair reversed-phase high performance liquid chromatography method for the quantification of isoaspartic acid in a monoclonal antibody

Cited by 3 publications

References 34 publications

Full Antibody Primary Structure and Microvariant Characterization in a Single Injection Using Transient Isotachophoresis and Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry

Full Antibody Primary Structure and Microvariant Characterization in a Single Injection Using Transient Isotachophoresis and Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry

Technological Platforms to Study Plant Lipidomics

Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy

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