RESUMO
MAB007, an IgG1 monoclonal antibody, is unique because of the presence of a free cysteine residue in the Fab region at position 104 on the heavy chain in the CDR3 region. Mass spectrometric analysis of intact MAB007 showed multiple peaks varying in mass by 120-140 Da that cannot be fully attributed to glycosylation isoforms typically present in IgG molecules. Limited proteolysis of MAB007 with Lys-C led to a single cleavage at the C-terminus of a lysine residue in the hinge region of the heavy chain at position 222, generating free Fab and Fc fragments. Reversed-phase liquid chromatography/mass spectrometry analysis of the Fab and Fc fragments revealed several modifications. The Fab fraction showed cysteinylation of a free cysteine in the CDR3 region resulting in a mass shift of 119 Da. Using limited proteolysis, we also identified modifications resulting in a mass increase of 127 Da in the Fc region, corresponding to C-terminal lysine variants in the heavy chain. Other modifications, such as oxidation (+16 Da) and succinimide formation (-17 Da), were also detected in the Fab fragment. The cysteinylation observed after limited proteolysis was confirmed by peptide mapping coupled with tandem mass spectrometry analysis.
Assuntos
Anticorpos Monoclonais/análise , Cisteína/análise , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Lisina/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Cisteína/química , Cromatografia Gasosa-Espectrometria de Massas , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/metabolismo , Mapeamento de Peptídeos , Isoformas de Proteínas , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
PURPOSE: The aim of the study is to provide a methodology for assigning unpaired cysteine residues in proteins formulated in a variety of different conditions to identify structural heterogeneity as a potential cause for protein degradation. METHODS: 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was employed for cyanylating free cysteines in proteins and peptides. Subsequent basic cleavage of the peptide bond at the N-terminal side of the cyanylated cysteines provided direct information about their location. RESULTS: CDAP was successfully employed to a wide variety of labeling conditions. CDAP was reactive between pH 2.0 and 8.0 with a maximum labeling efficiency at pH 5.0. Its reactivity was not affected by excipients, salt or denaturant. Storing CDAP in an organic solvent increased its intrinsic stability. It was demonstrated that CDAP can be employed as a thiol-directed probe to investigate structural heterogeneity of proteins by examining the accessibility of unpaired cysteine residues. CONCLUSION: CDAP is a unique cysteine-labeling reagent because it is reactive under acidic conditions. This provides an advantage over other sulfhydryl labeling reagents as it avoids potential thiol-disulfide exchange. Optimization of the cyanylation reaction allowed the utilization of CDAP as a thiol-directed probe to investigate accessibility of sulfhydryl groups in proteins under various formulation conditions to monitor structural heterogeneity.
