title: Sequence-specific formation of d-amino acids in a monoclonal antibody during light exposure.
abstract: The photoirradiation of a monoclonal antibody 1 (mAb1) at λ = 254 nm and λmax = 305 nm resulted in the sequence-specific generation of d-Val, d-Tyr, and potentially d-Ala and d-Arg, in the heavy chain sequence [95-101] YCARVVY. d-Amino acid formation is most likely the product of reversible intermediary carbon-centered radical formation at the (α)C-positions of the respective amino acids ((α)C(•) radicals) through the action of Cys thiyl radicals (CysS(•)). The latter can be generated photochemically either through direct homolysis of cystine or through photoinduced electron transfer from Trp and/or Tyr residues. The potential of mAb1 sequences to undergo epimerization was first evaluated through covalent H/D exchange during photoirradiation in D2O, and proteolytic peptides exhibiting deuterium incorporation were monitored by HPLC-MS/MS analysis. Subsequently, mAb1 was photoirradiated in H2O, and peptides, for which deuterium incorporation in D2O had been documented, were purified by HPLC and subjected to hydrolysis and amino acid analysis. Importantly, not all peptide sequences which incorporated deuterium during photoirradiation in D2O also exhibited photoinduced d-amino acid formation. For example, the heavy chain sequence [12-18] VQPGGSL showed significant deuterium incorporation during photoirradiation in D2O, but no photoinduced formation of d-amino acids was detected. Instead this sequence contained ca. 22% d-Val in both a photoirradiated and a control sample. This observation could indicate that d-Val may have been generated either during production and/or storage or during sample preparation. While sample preparation did not lead to the formation of d-Val or other d-amino acids in the control sample for the heavy chain sequence [95-101] YCARVVY, we may have to consider that during hydrolysis N-terminal residues (such as in VQPGGSL) may be more prone to epimerization. We conclude that the photoinduced, radical-dependent formation of d-amino acids requires not only the intermediary formation of a (α)C(•) radical but also sufficient flexibility of the protein domain to allow both pro-chiral faces of the (α)C(•) radical to accept a hydrogen atom.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas , 2095 Constant Avenue, Lawrence, Kansas 66047, United States.
authors.forename: Olivier
authors.initials: O
authors.lastname: Mozziconacci
authors.forename: Christian
authors.initials: C
authors.lastname: Schöneich
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000528:radiation effects; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000528:radiation effects; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D006860:Hydrogen Bonding; D008027:Light; D010446:Peptide Fragments / Q000737:chemistry* / Q000528:radiation effects; D010782:Photolysis; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D004220:Disulfides; D010446:Peptide Fragments; D006859:Hydrogen; D003903:Deuterium; D003545:Cysteine
keywords: IgG1; d-amino acids; photodegradation; thiyl radicals; tryptophan