Photodisruption of the Structurally Conserved Cys-Cys-Trp Triads Leads to Reduction-Resistant Scrambled Intrachain Disulfides in an IgG1 Monoclonal Antibody

Abstract

Photostability conditions as prescribed by ICH guidelines induced highly reduction-resistant scrambled disulfides that contribute to the population of apparent nonreducible aggregates in an IgG1 mAb. Photoinduced cross-linked species were isolated under reducing conditions using an organic phase size exclusion chromatography (OP-SEC) method, followed by O-labeling tryptic mapping to identify cross-linked peptides. Disulfide scrambling was observed within the IgG1 structurally conserved-intrachain cysteine-cysteine-tryptophan triads (Cys-Cys-Trp), and correlated with Trp-to-kynurenine (Kyn) photodegradation within these triads. We hypothesize that intrachain disulfides protect the proximal Trp within the Cys-Cys-Trp triads from photodegradation by enabling dissipation of Trp-absorbed UV energy via electron transfer to the disulfide bond. Finally, we propose three distinct mechanisms of photochemical degradation of monoclonal antibodies mediated by Trp residues.

Unique Impacts of Methionine Oxidation, Tryptophan Oxidation, and Asparagine Deamidation on Antibody Stability and Aggregation

“…The fact that these crosslinks (which primarily form for mAb 1) are nonreducible suggests mechanisms other than disulfide scrambling, although cases of the formation of reduction-resistant disulfide bonds through disulfide scrambling have been reported. 20 It is notable that previous studies have identified similar types of nonreducible crosslinks for mAbs incubated at similar conditions as those used in our study for promoting deamidation, namely elevated temperature (e.g., 37 C-45 C) and high pH (pH 8-10). 40,[57][58][59][60] Moreover, the observed degree of crosslink formation was significantly different for different antibodies, 57 as we observed in our study.…”

“…It is also notable that previous studies of photo-oxidation of antibodies found that disulfide bond shuffling can occur via oxidation of Trp residues located in close proximity to intramolecular disulfide bonds through electron transfer or singlet oxygen pathways, and such disulfide scrambling also promotes antibody aggregation. 20,21,48 However, whether similar or unique mechanisms mediate disulfide scrambling for chemical oxidation and photo-oxidation of antibodies is unclear and requires more detailed studies in the future. It is also important to consider that the aggregation behavior of mAbs is strongly influenced by the type of stress and the specific formulation conditions.…”

“…One such mechanism is that these chemical modifications can lead to the formation of covalently cross-linked species between different antibody domains. 20,[39][40][41] Therefore, we used SDS-PAGE to evaluate the formation of reducible and nonreducible cross-linked species of the control, oxidized, and deamidated mAbs (Fig. 5).…”

“…16 Tryptophan (Trp) is also particularly susceptible to oxidation, and oxidation of Trp residues in antibody complementarity-determining regions (CDRs) has been linked to loss of antigen binding. [17][18][19] Moreover, Trp oxidation has been linked to reduced antibody physical stability by promoting disulfide scrambling 20,21 and aggregation. 17,22,23 Asparagine (Asn) deamidation is another common type of antibody chemical modification, which has been linked to reduced affinity due to deamidation of CDR residues [24][25][26] as well as variable impacts on antibody conformational stability 24,27,28 and aggregation.…”

Direct Ultraviolet Laser-Induced Reduction of Disulfide Bonds in Insulin and Vasopressin

“…[3][4][5][6][7]9 The hypothesis has been used more recently to explain disulfide bond scrambling (formation of non-native disulfide bonds) by reoxidation of photogenerated thiols in human growth hormone and IgG1 antibody. 10,11 UV light has been used to excite solution mixtures of acetone and alcohol to activate radical chemistry that cleaves disulfide bonds into reduced and alkylated sulfur atoms in specific ratios depending on the alcohol. 12 In the gas phase, ultraviolet photodissociation (UVPD) of the covalent bonds in peptides and proteins can be used as a fragmentation method in tandem mass spectrometry.…”