Middle-up characterization of monoclonal antibodies by online reduction liquid chromatography-mass spectrometry

This study describes the fully automated middle-up characterization of monoclonal antibodies (mAbs) and next-generation variants by online reduction liquid chromatography-mass spectrometry (LC-MS). Proteins were trapped on-column and subjected to online desalting, denaturation and reduction prior to reversed phase elution of the created subunits in the MS. The evaluation of more than 20 different therapeutic proteins including full length mAbs (subclasses IgG1, IgG2 and IgG4), bispecific antibodies, antibody fragments, fusion proteins and antibody-drug conjugates (ADC) revealed that the online reduction method is as powerful as the widely applied offline sample preparation with dithiothreitol (DTT) as reducing agent and guanidine hydrochloride (Gnd.HCl) as denaturant and tackles some major disadvantages associated with the latter method, i.e. corrosion of stainless steel components, adduct formation impacting spectral quality and sample stability. The value of the online reduction LC-MS method is also enforced by its ability to reveal unstable antibody variants such as succinimide intermediates of asparagine deamidation and aspartic acid isomerization which are often lost when using the offline sample preparation method. The performance of the online reduction LC-MS set-up was verified and it was revealed that the method is precise with RSD values below 0.25% and 3.0% for retention time and area, respectively. Carry-over is within acceptable limits (< 0.5%) and the reducing buffer is stable up to 24 hours.