Session: Session 2

Multi-Identification, Characterization, and Quantification of Related Substances by 1D SEC-UV and 2D RP-MS/HRMS Heart Cutting on Biotherapeutics (mAb)

The development of biotherapeutics is revolutionizing human health. However, these pharmaceutical products can produce related substances during their development, transport, and storage. It is therefore important to predict the appearance of these related substances through forced degradation or stability studies. Stress conditions can be employed to generate related substances likely to emerge in the final product. Various liquid chromatography methods (RP/SEC/IEX-UV) can be used to detect and quantify related substances, which often cannot be directly identified and characterized using mass spectrometry due to the presence of non-volatile salts in mobile phases. 2D-LC allows the use of a 1D for UV quantification with a validated GMP method that is not MS-compatible, and a second dimension that is MS-compatible. However, for each related substance analyzed, an injection is performed, which can be a considerable waste of time. The aim of this study was to develop a 2D method adaptable to a first dimension imposed with LC-MS analysis for multiple identification.



This study was carried out on a UPLC vanquish chromatographic chain using two column compartments with 6 valves, coupled to an Exploris 240. A reference mAb was used to obtain related substances after forced degradation. The 1D uses an SEC with Sodium Phosphate. An optimization was carried out for the second dimension, choice of column, gradient, valves…



The method for identifying and characterizing up to 6 related substances in a single injection, using a 6-loop heart cutting approach. A phenyl-type column was selected for the second dimension with rapid elution from 20% to 50% acetonitrile. 200 to 20µL loops were used depending on the time of impurity peaks.



This study has enabled the development of a rapid multi-identification and characterization method for biotherapeutic products. which saves around 2 out of 3 hours for a sample of biological product, and also reduces analysis costs.