title: Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate in Vivo Deamidation of Therapeutic Antibodies.
abstract: Deamidation of therapeutic antibodies may result in decreased drug activity and undesirable changes in pharmacokinetics and immunogenicity. Therefore, it is necessary to monitor the deamidation levels [during storage] and after in vivo administration. Because of the complexity of in vivo samples, immuno-affinity capture is widely used for specific enrichment of the target antibody prior to LC-MS. However, the conventional use of bead-based methods requires large sample volumes and extensive processing steps. Furthermore, with automation difficulties and extended sample preparation time, bead-based approaches may increase artificial deamidation. To overcome these challenges, we developed an automated platform to perform tip-based affinity capture of antibodies from complex matrixes with rapid digestion and peptide elution into 96-well microtiter plates followed by LC-MS analysis. Detailed analyses showed that the new method presents high repeatability and reproducibility with both intra and inter assay CVs < 8%. Using the automated platform, we successfully quantified the levels of deamidation of a humanized monoclonal antibody in cynomolgus monkeys over a time period of 12 weeks after administration. Moreover, we found that deamidation kinetics between in vivo samples and samples stressed in vitro at neutral pH were consistent, suggesting that the in vitro stress test may be used as a method to predict the liability to deamidation of therapeutic antibodies in vivo.
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: John C
authors: JC
authors: Tran
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Daniel
authors: D
authors: Tran
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Amy
authors: A
authors: Hilderbrand
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Nisana
authors: N
authors: Andersen
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Tao
authors: T
authors: Huang
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Karin
authors: K
authors: Reif
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Isidro
authors: I
authors: Hotzel
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Eric G
authors: EG
authors: Stefanich
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Yichin
authors: Y
authors: Liu
authors: Biochemical and Cellular Pharmacology, ‡Protein Analytical Chemistry, §Immunology, ∥Antibody Engineering, and ⊥Preclinical and Translational Pharmacokinetics, Genentech, Inc. , South San Francisco, California 94080-4990, United States.
authors: Jianyong
authors: J
authors: Wang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism* / Q000627:therapeutic use; D001331:Automation; D016466:CHO Cells; D002853:Chromatography, Liquid; D003412:Cricetulus; D003641:Deamination; D004912:Erythrocytes; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008252:Macaca fascicularis; D013058:Mass Spectrometry
chemical_list: D000906:Antibodies