Improvement of Peak Integration in Capillary Electrophoresis: Reference Data Set No. 1.

Capillary electrophoresis (CE) often provides superior separation of macromolecules such as monoclonal antibodies (mAbs), a major biopharmaceutical class, compared with liquid chromatography. However, electropherograms frequently exhibit complex baselines and peak shapes that are not reliably handled by integration algorithms designed for chromatographic data, and manual integration is often required. Many concepts have been proposed to improve peak integration, ranging from incremental algorithmic refinements and signal-to-noise (S/N)-based approaches to artificial intelligence (AI)-driven strategies, but objective performance comparisons are not possible without shared reference data sets and agreed peak limits. To address this gap, we initiated a multinational collaboration involving industrial and academic laboratories to create a comprehensive reference data set for CE peak integration. A total of 227 challenging and practically relevant electropherograms were collected from diverse applications, converted to a standardized format, and independently integrated by multiple experts. Using dedicated software tools and a structured consensus process, mutually accepted reference integration limits were established for each data set. These reference electropherograms, together with the underlying integration rules, are now made available to the scientific community. Analysis of the reference data set identified general principles for reliable peak integration, including the importance of standardized zoom levels and consistent handling of small peaks near the noise level. The data set provides a common foundation for benchmarking commercial chromatography data systems (CDS) and for developing and validating new algorithmic and AI-based integration methods. We expect this work to speed up the development of practical, automated integration strategies for CE and that these core concepts can be applied to other separation techniques.