Targeted Bottom-up Characterization of Recombinant Monoclonal Antibodies by Multidimensional LC/MS

On-line bottom-up approaches have recently emerged as promising alternatives to standard off-line processes for characterizing post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs). The benefits of on-line processing include reductions in required sample amount and sample handling, as well as reducing the overall turnaround time. However, shortening digestion time for the on-line approach of an intact mAb can cause incomplete peptide cleavages, leading to low sequence coverage and poor repeatability of analyses. For the first time, we describe a novel, automated targeted bottom-up strategy consisting of reducing the complexity of intact mAb by digesting the product into small ∼25 kDa fragments, followed by an on-line peptide mapping analysis of each fragment. For this purpose, a four-dimensional-liquid chromatography/mass spectrometry (4D-LC/MS) method was developed using an immobilized IdeS-high-performance liquid chromatography (HPLC) column as a first dimension (¹D) for on-line digestion, followed by a (²D) on-column reversed-phase liquid chromatography (RPLC) for reduction and fragments separation. Then, only one fragment was selected for digestion using a (³D) immobilized trypsin cartridge and, finally, the obtained peptides were analyzed by (⁴D) RPLC-MS. This strategy considerably improved the on-line digestion efficiency with higher sequence coverages (LC and HC >97%), thus allowing various PTMs including oxidation, deamidation, and isomerization located in the complementarity-determining regions (CDRs), as well as N-glycans present on the Fc/2 fragment, to be monitored with similar sensitivity to those obtained with standard off-line approaches. Additional investigations at a middle-up level were also performed via a three-dimensional-LC/MS (3D-LC/MS) approach within the same system, demonstrating the feasibility to achieve a multilevel comprehensive characterization of mAbs.