Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography

Cao, Mingyan; Mel, Niluka De; Wang, Jihong; Parthemore, Conner; Jiao, Yang; Chen, Weimin; Lin, Shi-Hua; Liu, Dengfeng; Kilby, Greg W.; Chen, Xiaoyu

doi:10.1016/j.xphs.2021.09.006

Cited by 13 publications

(10 citation statements)

References 27 publications

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“…It is readily apparent that the attachment of the four drug-linkers to adalimumab results in a far more hydrophobic moiety. Similar levels of the common post-translational modification of cyclization of N-terminal glutamate to pyroglutamate 27 were observed in both adalimumab and ABBV-3373.…”

Section: ■ Results and Discussionsupporting

confidence: 57%

Optimization of Drug-Linker to Enable Long-term Storage of Antibody–Drug Conjugate for Subcutaneous Dosing

Hobson

Marvin

et al. 2023

J. Med. Chem.

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To facilitate subcutaneous dosing, biotherapeutics need to exhibit properties that enable high-concentration formulation and long-term stability in the formulation buffer. For antibody–drug conjugates (ADCs), the introduction of drug-linkers can lead to increased hydrophobicity and higher levels of aggregation, which are both detrimental to the properties required for subcutaneous dosing. Herein we show how the physicochemical properties of ADCs could be controlled through the drug-linker chemistry in combination with prodrug chemistry of the payload, and how optimization of these combinations could afford ADCs with significantly improved solution stability. Key to achieving this optimization is the use of an accelerated stress test performed in a minimal formulation buffer.

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Section: ■ Results and Discussionsupporting

confidence: 57%

Optimization of Drug-Linker to Enable Long-term Storage of Antibody–Drug Conjugate for Subcutaneous Dosing

Hobson

Marvin

et al. 2023

J. Med. Chem.

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“…All samples evaluated had binding affinities of ∼2 nM, and no significant differences in binding affinity or kinetics were determined due to the observed modifications (Table and Figure S5). We did not measure HER2 ECD binding for individual charge variants of pertuzumab, since modifications located in the Fc region and at the N-terminus of charge variants are not expected to affect antigen binding. ,, …”

Section: Resultsmentioning

confidence: 99%

“…We did not measure HER2 ECD binding for individual charge variants of pertuzumab, since modifications located in the Fc region and at the N-terminus of charge variants are not expected to affect antigen binding. 12,38,39 Clinical Samples Analysis. LC−MS/MS peptide mapping analysis of plasma samples from patients treated with pertuzumab was performed after the targeted enrichment of pertuzumab and its charge variants from these samples using specific Affimers 22 (Table 5).…”

Section: ■ Resultsmentioning

confidence: 99%

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Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation

et al. 2023

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Pertuzumab is a monoclonal antibody used for the treatment of HER2-positive breast cancer in combination with trastuzumab. Charge variants of trastuzumab have been extensively described in the literature; however, little is known about the charge heterogeneity of pertuzumab. Here, changes in the ionexchange profile of pertuzumab were evaluated by pH gradient cation-exchange chromatography after stressing it for up to 3 weeks at physiological and elevated pH and 37 °C. Isolated charge variants arising under stress conditions were characterized by peptide mapping. The results of peptide mapping showed that deamidation in the Fc domain and N-terminal pyroglutamate formation in the heavy chain are the main contributors to charge heterogeneity. The heavy chain CDR2, which is the only CDR containing asparagine residues, was quite resistant to deamidation under stress conditions according to peptide mapping results. Using surface plasmon resonance, it was shown that the affinity of pertuzumab for the HER2 target receptor does not change under stress conditions. Peptide mapping analysis of clinical samples showed an average of 2−3% deamidation in the heavy chain CDR2, 20−25% deamidation in the Fc domain, and 10−15% N-terminal pyroglutamate formation in the heavy chain. These findings suggest that in vitro stress studies are able to predict in vivo modifications.

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“…In recent years, there has been a significant increase in the literature investigating pyroglutamination in vitro, mainly due to its frequency in bottom-up proteomics experiments. ,,, This conversion not only changes the primary structure of the peptide but also impacts the physicochemical properties of the solution components. The Q → Pyr conversion eliminates the positive charge of the N-terminus, leading to the formation of a more hydrophobic and insoluble final product. , Such transformations significantly impact the physicochemical characteristics of peptides, with implications for the biological functions of these species.…”

Section: Introductionmentioning

confidence: 99%

Pyroglutamination-Induced Changes in the Physicochemical Features of a CXCR4 Chemokine Peptide: Kinetic and Structural Analysis

et al. 2023

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We investigate the physicochemical effects of pyroglutamination on the QHALTSV-NH2 peptide, a segment of cytosolic helix 8 of the human C–X–C chemokine G-protein-coupled receptor type 4 (CXCR4). This modification, resulting from the spontaneous conversion of glutamine to pyroglutamic acid, has significant impacts on the physicochemical features of peptides. Using a static approach, we compared the transformation in different conditions and experimentally found that the rate of product formation increases with temperature, underscoring the need for caution during laboratory experiments to prevent glutamine cyclization. Circular dichroism experiments revealed that the QHALTSV-NH2 segment plays a minor role in the structuration of H8 CXCR4; however, its pyroglutaminated analogue interacts differently with its chemical environment, showing increased susceptibility to solvent variations compared to the native form. The pyroglutaminated analogue exhibits altered behavior when interacting with lipid models, suggesting a significant impact on its interaction with cell membranes. A unique combination of atomic force microscopy and infrared nanospectroscopy revealed that pyroglutamination affects supramolecular self-assembly, leading to highly packed molecular arrangements and a crystalline structure. Moreover, the presence of pyroglumatic acid has been found to favor the formation of amyloidogenic aggregates. Our findings emphasize the importance of considering pyroglutamination in peptide synthesis and proteomics and its potential significance in amyloidosis.

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Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography

Cited by 13 publications

References 27 publications

Optimization of Drug-Linker to Enable Long-term Storage of Antibody–Drug Conjugate for Subcutaneous Dosing

Optimization of Drug-Linker to Enable Long-term Storage of Antibody–Drug Conjugate for Subcutaneous Dosing

Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation

Pyroglutamination-Induced Changes in the Physicochemical Features of a CXCR4 Chemokine Peptide: Kinetic and Structural Analysis

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