Automated On-tip Affinity Capture Coupled with Mass Spectrometry to Characterize Intact Antibody-Drug Conjugates from Blood
| Autor: | Neha Srikumar, Aimee Fourie-O'Donohue, Yichin Liu, John C. Tran, Phillip Y. Chu, Katherine R. Kozak, Ke Sherry Li |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Antibody-drug conjugate Immunoconjugates Chromatography Chemistry Elution Antibodies Monoclonal Haplorhini Repeatability Mass spectrometry Proteomics Mass Spectrometry In vitro Rats 03 medical and health sciences 030104 developmental biology Structural Biology In vivo Animals Humans Spectroscopy Chromatography Liquid Conjugate |
| Zdroj: | Journal of the American Society for Mass Spectrometry. 29:1532-1537 |
| ISSN: | 1044-0305 |
| Popis: | Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method. Graphical Abstract ᅟ. |
| Databáze: | OpenAIRE |
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