Rapid Distinction of Leucine and Isoleucine in Monoclonal Antibodies Using Nanoflow LCMSⁿ

Monoclonal antibodies (mAbs) are large heterogeneous molecules that represent a growing class of therapeutics. De novo sequencing of mAbs becomes necessary when the original cell line or the cDNA is unavailable. An important feature in sequencing of mAbs is the discrimination of isobaric residues (Xle): leucine (Leu) and isoleucine (Ile). An incorrect identification of the Xle site, especially in the complementarity determining regions (CDRs), can result in the production of an antibody with severely compromised efficacy. Multistage fragmentation (MSⁿ) in the mass spectrometer can provide sufficient evidence for Ile/Leu discrimination. However, most existing methods utilize direct infusion of purified peptides, demanding peptide enrichment which can be labor-intensive and requires large amount of material. Here we introduce an online nano-LCMSⁿ method, which depending on the nature of the peptide, exploits either generation of a signature 69 Da ion from Ile or formation of unique w-ions employing MS³ (ETD-HCD) for rapid Ile/Leu distinction. This reliable and sensitive method utilizes the Orbitrap Fusion tribid mass spectrometer to rapidly assign multiple Xle residues in the CDRs of mAbs.