Rapid purity and charge-variant analysis of bispecific antibodies using microfluidic capillary electrophoresis-mass spectrometry
- 1Protein Purification & Characterization Department, Polpharma Biologics Utrecht, Yalelaan 46, 3584 CM Utrecht, The Netherlands
- 2Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Amsterdam Institute for Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
- 3Centre for Analytical Sciences Amsterdam (CASA), Amsterdam, The Netherlands
Abstract
Bispecific antibody (BsAb) therapeutics have the ability to bind two targets simultaneously, which opens up new pathways for disease treatment. BsAbs consist of distinct heavy and light chains that need to be assembled correctly to form the appropriate heterodimer. However, during development and production, misassembly of chains may occur, which affects BsAb purity, and therefore needs to be monitored. Here, we demonstrate the usefulness of microfluidic capillary electrophoresis-mass spectrometry (MCE-MS) for rapid purity and proteoform profiling of BsAb samples. MCE was capable of resolving homodimeric antibody species as well as charge variants from a model BsAb, while MS detection provided identification of these BsAb charge variants and glycoforms. A low-resolution MCE-MS method allowed quantitative determination of homodimer content in less than 3 min, whereas a high-resolution MCE-MS method delivered the detailed charge-variant and glycoform profile of the BsAb therapeutic within 15 min. We applied both methods in tandem for clone screening and downstream purification monitoring of large biosimilar BsAb sample sets. First, the homodimer-heterodimer ratio of BsAb cell line samples and purification process intermediates was established using the fast MCE-MS method. Next, based on the determined BsAb purity, samples of interest were selected and analyzed with the high-resolution MCE-MS method yielding a quality profile, permitting rational clone selection and optimization of the purification process. Overall, the developed MCE-MS workflow exhibits great potential to accelerate and enhance decision-making during BsAb development and production.
Graphical Abstract:
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s00216-026-06748-z.
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BibTeX
@article{cageling2026rapid,
title = {{Rapid purity and charge-variant analysis of bispecific antibodies using microfluidic capillary electrophoresis-mass spectrometry}},
author = {Ruben Cageling and Karin Lubbers-Geuijen and Govert W. Somsen and Kevin Jooß},
journal = {Analytical and Bioanalytical Chemistry},
year = {2026},
month = aug,
doi = {10.1007/s00216-026-06748-z},
url = {https://doi.org/10.1007/s00216-026-06748-z},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Rapid purity and charge-variant analysis of bispecific antibodies using microfluidic capillary electrophoresis-mass spectrometry
AU - Ruben Cageling
AU - Karin Lubbers-Geuijen
AU - Govert W. Somsen
AU - Kevin Jooß
PY - 2026
DA - 2026/08/22
JO - Analytical and Bioanalytical Chemistry
DO - 10.1007/s00216-026-06748-z
UR - https://doi.org/10.1007/s00216-026-06748-z
AB - Bispecific antibody (BsAb) therapeutics have the ability to bind two targets simultaneously, which opens up new pathways for disease treatment. BsAbs consist of distinct heavy and light chains that need to be assembled correctly to form the appropriate heterodimer. However, during development and production, misassembly of chains may occur, which affects BsAb purity, and therefore needs to be monitored. Here, we demonstrate the usefulness of microfluidic capillary electrophoresis-mass spectrometry (MCE-MS) for rapid purity and proteoform profiling of BsAb samples. MCE was capable of resolving homodimeric antibody species as well as charge variants from a model BsAb, while MS detection provided identification of these BsAb charge variants and glycoforms. A low-resolution MCE-MS method allowed quantitative determination of homodimer content in less than 3 min, whereas a high-resolution MCE-MS method delivered the detailed charge-variant and glycoform profile of the BsAb therapeutic within 15 min. We applied both methods in tandem for clone screening and downstream purification monitoring of large biosimilar BsAb sample sets. First, the homodimer-heterodimer ratio of BsAb cell line samples and purification process intermediates was established using the fast MCE-MS method. Next, based on the determined BsAb purity, samples of interest were selected and analyzed with the high-resolution MCE-MS method yielding a quality profile, permitting rational clone selection and optimization of the purification process. Overall, the developed MCE-MS workflow exhibits great potential to accelerate and enhance decision-making during BsAb development and production. Graphical Abstract: Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s00216-026-06748-z.
SN - 1618-2642
SN - 1618-2650
ER -