Comprehensive Multidimensional Liquid Chromatography–Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody

Journal of the American Society for Mass Spectrometry, vol. 33, pp. 2319–2327

Abstract

Identification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel antibody formats. As a standard analytical approach, manual offline fractionation of charge variants by cation-exchange chromatography followed by comprehensive analytical testing is applied. These conventional workflows are time-consuming and labor-intensive and overall reach their limits in terms of chromatographic separation of enhanced structural heterogeneities raised from new antibody formats. For these reasons, we aimed to develop an alternative online characterization strategy for charge variant characterization of a therapeutic bispecific antibody by online mD-LC-MS at middle-up (2D-LC-MS) and bottom-up (4D-LC-MS) level. Using the implemented online mD-LC-MS approach, all medium- and even low-abundant product variants previously identified by offline fraction experiments and liquid chromatography mass spectrometry could be monitored. The herein reported automated online mD-LC-MS methodology therefore represents a complementary and in part alternative approach for analytical method validation including multiattribute monitoring (MAM) strategies by mass spectrometry, offering various benefits including increased throughput and reduced sample handling and combined protein information at intact protein and peptide level.

Authors 9

1. Ingrid Grunert corresponding
   Affiliation as printed

   Pharma Technical Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany

2. Katrin Heinrich
   Affiliation as printed

   Pharma Technical Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany

3. Michael Hingar
   Affiliation as printed

   Pharma Technical Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany

4. Juliane Ernst
   Affiliation as printed

   Pharma Technical Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany

5. Katrin Bomans
   Affiliation as printed

   Pharma Technical Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany

6. Arnaud Fevre

   Roche (Switzerland)

   Affiliation as printed

   Pharma Technical Development, Hoffmann-La Roche, 4070 Basel, Switzerland

7. Dietmar Reusch
   Affiliation as printed

   Pharma Technical Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany

8. Manfred Wuhrer Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 Leiden, The Netherlands

9. Patrick Bulau

   Roche (Switzerland)

   Affiliation as printed

   Pharma Technical Development, Hoffmann-La Roche, 4070 Basel, Switzerland