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.

Thiyl radicals and induction of protein degradation

“…In fact, D-alanine formation was detected in model peptides [60], and during light-induced thiyl radical generation in IgG1[88]. These observations are consistent with synthetic applications, where thiyl radicals where used for the racemization of amines [89].…”

Protein thiyl radical reactions and product formation: a kinetic simulation

“…However, this does not imply quantitative repair . Our model suggests the formation of additional products, mainly protein aldehydes from the hydrolysis of thioaldehydes and protein-bound D-cysteine (or other D-amino acids[51]). Such species would not be found in standard assays.…”

“…While thiyl radicals were not generated through physiological processes in these studies, they are relevant to physiology as they demonstrate what can happen when thiyl radicals are generated on proteins. In the absence of oxygen, the reversible generation of carbon-centered radicals has led to the conversion of L-Ala to D-Ala in a model peptide[32], and to the sequence-specific generation of D-amino acids on IgG1[51]. Hence, thiyl radical generation on proteins is a viable source for D-amino acid generation, and such reactions have to be taken into account when simulating thiyl radical reaction pathways in proteins (see below).…”

The Effects of Light-Accelerated Degradation on the Aggregation of Marketed Therapeutic Monoclonal Antibodies Evaluated by Size-Exclusion Chromatography With Diode Array Detection

“…In particular, the sequence-specific generation of D-Val, D-Tyr, and potentially D-Ala and D-Arg was proposed. 21 Recent research also demonstrated that exposing mAbs to light during their production process clearly alters the charge variants profile of the final product. 22 There is therefore extensive evidence that photodegradation leads primarily to chemical changes in single amino acids of proteins and that trp, tyr, phe, and cys are the primary targets of photodegradation.…”