title: Screening methods to identify indole derivatives that protect against reactive oxygen species induced tryptophan oxidation in proteins.
abstract: Oxidative damage to proteins is one of the most prominent chemical degradation pathways that are of concern for drug product development in the biotechnology industry. Especially susceptible to oxidation are the Met and Trp residues in proteins. While L-Met and L-Trp have been shown to act as antioxidants typically protecting proteins against Met and Trp oxidation, respectively, l-Trp has been shown to be particularly sensitive to light, thereby producing various reactive oxygen species (ROS), including H2O2. There is hence a need to identify nonphotosensitive molecules that can protect Trp oxidation in proteins so that they can be easily handled under drug product manufacturing conditions. A combination of screening methods, namely, cyclic voltammetry (CV) and hydrogen peroxide generation upon photoirradiation, was used to screen several molecules to identify compounds that can act as antioxidants. Specifically, indole and tryptophan with hydroxy groups on the six-membered aromatic ring were found to have lower oxidation potentials than the parent compounds and produced the least amount of H2O2 upon light exposure. These derivatives were also found to sufficiently protect tryptophan oxidation in mAb1 against a variety of reactive oxygen species such as alkyl peroxides, hydroxyl radicals, and singlet oxygen and may be useful as part of the formulation toolkit to protect against protein degradation via oxidation.
authors.affiliation: Late Stage Pharmaceutical Development, Genentech, Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Parbir
authors.initials: P
authors.lastname: Grewal
authors.forename: Mary
authors.initials: M
authors.lastname: Mallaney
authors.forename: Kimberly
authors.initials: K
authors.lastname: Lau
authors.forename: Alavattam
authors.initials: A
authors.lastname: Sreedhara
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D007211:Indoles / Q000494:pharmacology*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D007211:Indoles; D017382:Reactive Oxygen Species; D014364:Tryptophan