title: Rapid Distinction of Leucine and Isoleucine in Monoclonal Antibodies Using Nanoflow LCMSn.
abstract: 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-LCMSnmethod, 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.
authors.affiliation: Discovery Attribute Sciences, Amgen , South San Francisco, California 94080, United States.
authors.forename: Dhanashri
authors.identifier: 0000-0003-1262-231X
authors.initials: D
authors.lastname: Bagal
authors.affiliation: Discovery Attribute Sciences, Amgen , South San Francisco, California 94080, United States.
authors.forename: Eddie
authors.initials: E
authors.lastname: Kast
authors.affiliation: Discovery Attribute Sciences, Amgen , South San Francisco, California 94080, United States.
authors.forename: Ping
authors.initials: P
authors.lastname: Cao
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D007532:Isoleucine / Q000032:analysis*; D007930:Leucine / Q000032:analysis*; D015394:Molecular Structure; D036103:Nanotechnology; D053719:Tandem Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D007532:Isoleucine; D007930:Leucine