Systematic analytical workflow for characterisation and identification of partially reduced species in monoclonal...

Fragmentation is a major degradation pathway ubiquitous to all therapeutic monoclonal antibody (mAb) and therefore, monitored throughout the manufacturing process. Here, we describe a three-step approach to 1) detect, 2) confirm and 3) characterize partially reduced fragment species in an immunoglobulin G1 (IgG1) mAb with prolonged hold time of harvested cell culture fluid (HCCF). Microchip capillary electrophoresis (MCE) and high-performance size exclusion chromatography (HPSEC) were used as fast and efficient screening methods to detect fragmentation. HPSEC was found to be underestimating fragmentation levels. To confirm and characterize the fragments, capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) was employed. Interestingly, the absence of fragments in the reduced CE-SDS analysis suggested partial reduction of disulphide bonds contributing to fragmentation in this mAb lot. This was further confirmed using reverse phase high performance liquid chromatography (RP-HPLC) coupled with mass spectrometry, which established the presence of heavy-heavy-light (HHL), heavy-heavy (HH), light-light dimer (LL), light chain (LC) and half antibody (HL) fragments with good mass accuracy. In this study, we demonstrated a readily applicable systematic strategy to support process development and investigate anomalous events in manufacturing. An additional highlight of this work is the data-driven comprehensive comparison of modern and conventional analytical techniques for fragment analysis.