Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug Conjugate
Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody–drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR),...
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| Format: | Article |
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MDPI AG
2023-10-01
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| Series: | Antibodies |
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| Online Access: | https://www.mdpi.com/2073-4468/12/4/66 |
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| author | Yue Huang Jiaqi Yuan Ruipeng Mu Robert J. Kubiak Kathryn Ball Mingyan Cao G. Patrick Hussmann Niluka de Mel Dengfeng Liu Lorin K. Roskos Meina Liang Anton I. Rosenbaum |
| author_facet | Yue Huang Jiaqi Yuan Ruipeng Mu Robert J. Kubiak Kathryn Ball Mingyan Cao G. Patrick Hussmann Niluka de Mel Dengfeng Liu Lorin K. Roskos Meina Liang Anton I. Rosenbaum |
| author_sort | Yue Huang |
| collection | DOAJ |
| description | Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody–drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography–tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology. |
| first_indexed | 2024-03-08T21:02:50Z |
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| id | doaj.art-b0bf267e4c2342369ff9562c70aa3c06 |
| institution | Directory Open Access Journal |
| issn | 2073-4468 |
| language | English |
| last_indexed | 2024-03-08T21:02:50Z |
| publishDate | 2023-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibodies |
| spelling | doaj.art-b0bf267e4c2342369ff9562c70aa3c062023-12-22T13:48:09ZengMDPI AGAntibodies2073-44682023-10-011246610.3390/antib12040066Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug ConjugateYue Huang0Jiaqi Yuan1Ruipeng Mu2Robert J. Kubiak3Kathryn Ball4Mingyan Cao5G. Patrick Hussmann6Niluka de Mel7Dengfeng Liu8Lorin K. Roskos9Meina Liang10Anton I. Rosenbaum11Integrated Bioanalysis, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, 121 Oyster Point Boulevard, South San Francisco, CA 94080, USAIntegrated Bioanalysis, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, 121 Oyster Point Boulevard, South San Francisco, CA 94080, USAIntegrated Bioanalysis, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, 121 Oyster Point Boulevard, South San Francisco, CA 94080, USAClinical Pharmacology and Quantitative Pharmacology, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USAClinical Pharmacology and Quantitative Pharmacology, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Granta Park, Cambridge CB21 6GH, UKDepartment of Analytical Sciences, Biopharmaceutical Development, R&D, AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USADepartment of Analytical Sciences, Biopharmaceutical Development, R&D, AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USADepartment of Analytical Sciences, Biopharmaceutical Development, R&D, AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USADepartment of Analytical Sciences, Biopharmaceutical Development, R&D, AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USAClinical Pharmacology and Quantitative Pharmacology, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USAIntegrated Bioanalysis, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, 121 Oyster Point Boulevard, South San Francisco, CA 94080, USAIntegrated Bioanalysis, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, 121 Oyster Point Boulevard, South San Francisco, CA 94080, USADeamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody–drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography–tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.https://www.mdpi.com/2073-4468/12/4/66deamidationhybrid liquid chromatography–mass spectrometry (LC-MS) assaypost-translational modification (PTM)biotransformationantibody–drug conjugate (ADC) |
| spellingShingle | Yue Huang Jiaqi Yuan Ruipeng Mu Robert J. Kubiak Kathryn Ball Mingyan Cao G. Patrick Hussmann Niluka de Mel Dengfeng Liu Lorin K. Roskos Meina Liang Anton I. Rosenbaum Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug Conjugate Antibodies deamidation hybrid liquid chromatography–mass spectrometry (LC-MS) assay post-translational modification (PTM) biotransformation antibody–drug conjugate (ADC) |
| title | Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug Conjugate |
| title_full | Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug Conjugate |
| title_fullStr | Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug Conjugate |
| title_full_unstemmed | Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug Conjugate |
| title_short | Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody–Drug Conjugate |
| title_sort | multiplex bioanalytical methods for comprehensive characterization and quantification of the unique complementarity determining region deamidation of medi7247 an anti asct2 pyrrolobenzodiazepine antibody drug conjugate |
| topic | deamidation hybrid liquid chromatography–mass spectrometry (LC-MS) assay post-translational modification (PTM) biotransformation antibody–drug conjugate (ADC) |
| url | https://www.mdpi.com/2073-4468/12/4/66 |
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