title: Liquid chromatography-fluorescence and liquid chromatography-mass spectrometry detection of tryptophan degradation products of a recombinant monoclonal antibody.
abstract: Light exposure is one of several conditions used to study the degradation pathways of recombinant monoclonal antibodies. Tryptophan is of particular interest among the 20 amino acids because it is the most photosensitive. Tryptophan degradation forms several products, including an even stronger photosensitizer and several reactive oxygen species. The current study reports a specific peptide mapping procedure to monitor tryptophan degradation. Instead of monitoring peptides using UV 214 nm, fluorescence detection with an excitation wavelength of 295 nm and an emission wavelength of 350 nm was used to enable specific detection of tryptophan-containing peptides. Peaks that decreased in area over time are likely to contain susceptible tryptophan residues. This observation can allow further liquid chromatography-mass spectrometry (LC-MS) analysis to focus only on those peaks to confirm tryptophan degradation products. After confirmation of tryptophan degradation, susceptibility of tryptophan residues can be compared based on the peak area decrease.
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Nowak
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Gomathinayagam
authors.initials: G
authors.lastname: Ponniah
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Guilong
authors.initials: G
authors.lastname: Cheng
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Adriana
authors.initials: A
authors.lastname: Kita
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Neill
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Yekaterina
authors.initials: Y
authors.lastname: Kori
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA. Electronic address: liuh@alxn.com.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014364:Tryptophan
keywords: Fluorescence detection; Mass spectrometry; Recombinant monoclonal antibody; Tryptophan