Validation of a high throughput fluorescent Capillary Electrophoresis Sodium Dodecyl Sulfate method for monoclonal antibody size heterogeneity assessment
- 1Agilent Technologies, Inc
Abstract
Fluorescent capillary gel electrophoresis (CGE) with sodium dodecyl sulfate (CE SDS) provides a powerful, high sensitivity alternative to ultraviolet (UV) based detection for characterizing therapeutic monoclonal antibodies (mAb). Regulatory and standards organizations, such as the United States Pharmacopeia (USP), include only UV based CE-SDS methods, hindering adoption of alternative detection methods. There is growing opportunity to expand beyond exclusively UV based CE SDS methods. In this study, we present an analytical validation of a light-emitting diode (LED) fluorescence-based parallel CE SDS method for both non reduced and reduced analysis of therapeutic antibodies. Using the NISTmAb reference material as a model system, size heterogeneity critical quality attributes (CQAs) including monomeric purity (non-reduced conditions), percent glycosylation (reduced conditions), and percent thioether (reduced conditions) were assessed. The fluorescence method demonstrated high specificity and precision with relative standard deviation (RSD) values <1% for monomeric purity and glycosylation, and <3% for thioether, as well as robust performance across variations in injection voltage, electrophoresis voltage, labeling temperature, and Labeling Buffer concentration. Ruggedness testing across users and reagent lots confirmed reproducibility, and accuracy assessments showed strong agreement with reported values from the National Institute of Standards (NIST) and traditional UV detection measurements. Linearity studies yielded coefficient of determination values R2>0.99 for both non reduced and reduced analyses. These results highlight the high sensitivity, stable baseline performance, and suitability of LED fluorescence-based parallel CE SDS as a validated, higher throughput alternative to traditional UV based methods for mAb quality control (QC).
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BibTeX
@misc{luttgeharm2026validation,
title = {{Validation of a high throughput fluorescent Capillary Electrophoresis Sodium Dodecyl Sulfate method for monoclonal antibody size heterogeneity assessment}},
author = {K. D. Luttgeharm and M. Grover and S.-Y. Huang and W. A. Pike},
howpublished = {bioRxiv},
year = {2026},
month = jul,
doi = {10.64898/2026.07.15.738750},
url = {https://www.biorxiv.org/content/10.64898/2026.07.15.738750v2},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - Validation of a high throughput fluorescent Capillary Electrophoresis Sodium Dodecyl Sulfate method for monoclonal antibody size heterogeneity assessment
AU - K. D. Luttgeharm
AU - M. Grover
AU - S.-Y. Huang
AU - W. A. Pike
PY - 2026
DA - 2026/07/15
PB - bioRxiv
DO - 10.64898/2026.07.15.738750
UR - https://www.biorxiv.org/content/10.64898/2026.07.15.738750v2
AB - Fluorescent capillary gel electrophoresis (CGE) with sodium dodecyl sulfate (CE SDS) provides a powerful, high sensitivity alternative to ultraviolet (UV) based detection for characterizing therapeutic monoclonal antibodies (mAb). Regulatory and standards organizations, such as the United States Pharmacopeia (USP), include only UV based CE-SDS methods, hindering adoption of alternative detection methods. There is growing opportunity to expand beyond exclusively UV based CE SDS methods. In this study, we present an analytical validation of a light-emitting diode (LED) fluorescence-based parallel CE SDS method for both non reduced and reduced analysis of therapeutic antibodies. Using the NISTmAb reference material as a model system, size heterogeneity critical quality attributes (CQAs) including monomeric purity (non-reduced conditions), percent glycosylation (reduced conditions), and percent thioether (reduced conditions) were assessed. The fluorescence method demonstrated high specificity and precision with relative standard deviation (RSD) values <1% for monomeric purity and glycosylation, and <3% for thioether, as well as robust performance across variations in injection voltage, electrophoresis voltage, labeling temperature, and Labeling Buffer concentration. Ruggedness testing across users and reagent lots confirmed reproducibility, and accuracy assessments showed strong agreement with reported values from the National Institute of Standards (NIST) and traditional UV detection measurements. Linearity studies yielded coefficient of determination values R2>0.99 for both non reduced and reduced analyses. These results highlight the high sensitivity, stable baseline performance, and suitability of LED fluorescence-based parallel CE SDS as a validated, higher throughput alternative to traditional UV based methods for mAb quality control (QC).
ER -