Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 1032, Issue -, Pages 91-102Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2016.06.049
Keywords
On-line comprehensive 2DLC; HICxRPLC-QTOF-MS; Quadrupole time-of-flight; Antibody-drug conjugate; ADCs; Odd DARs
Funding
- Swiss National Science Foundation [31003A 159494]
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This paper is the second part of a two-part series dedicated to the development of an on-line comprehensive HICxRPLC-UV/MS method for the characterization of a commercial inter-chain cysteine-linked ADC (brentuximab vedotin, Adcetris (R)). The first part focused on the optimization of the chromatographic conditions. In the second part of this series of papers, the structural characterization of the Brentuximab Vedotin was extensively discussed. With the combination of HIC and RPLC-MS data, the average DAR was easily measured in HIC and, at the same time, the predominant positional isomers were identified in RPLC-MS in one single injection. it was also demonstrated that the retention data obtained in the first and second dimensions was particularly useful to assist ADC characterization through the identification of sub-units. Using this methodology, the presence of odd DARs (1,3 and 5) arid their relative abundance was assessed by a systematic evaluation of HIC x RPLC-UV/MS data for both commercial and stressed ADC samples. Finally, once the exhaustive characterization of ADC was completed, MS could be conveniently replaced by UV detection to quickly assess the conformity of different ADCs batches. (C) 2016 Elsevier B.V. All rights reserved.
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