In that case, protein aggregates contain heterogeneous bityrosine cross-links and the abundance of tryptic digested peptides that contain a specific cross-link would be too low to be detected by mass spectroscopy

In that case, protein aggregates contain heterogeneous bityrosine cross-links and the abundance of tryptic digested peptides that contain a specific cross-link would be too low to be detected by mass spectroscopy. as strategies to minimize such aggregate formation during the development and storage of therapeutic proteins. strong class=”kwd-title” Keywords: monoclonal antibody, free radical, protein aggregation, oxidation, excipient 1. Introduction Oxidation-induced degradation of therapeutic proteins is commonly observed during pharmaceutical manufacturing, handling, and storage [1,2,3]. These oxidation reactions occur when there are activated oxygen species including singlet oxygen (1O2), superoxide radical (O2??), hydroxyl radical (?OH), and peroxide (-OO-) in the environment. Reactive oxygen species can be created through light, heat, free radicals, or transition metals [4,5]. These species can oxidize methionine (Met), tryptophan (Trp), histidine (His), tyrosine (Tyr), and cysteine (Cys) residues in proteins [6]. For example, it has been reported that ultraviolet (UV) irradiation induced oxidation of Met and Trp residues in a monoclonal antibody (mAb) [7], thermal or chemical stresses caused Met oxidation in proteins [8,9,10], and free radicals promoted Tyr oxidation in ATPase [11]. Oxidative modifications can alter proteins secondary and tertiary structures [12], hydrophobicity [13], stability [14], biological activity [14], and plasma circulation half-life [15]. Owing to these potential impacts, oxidation is typically an important quality attribute to be closely monitored during the development of therapeutic proteins. Various stress models including light and chemical stresses are often used to identify potential oxidation hot spots in therapeutic proteins and to characterize the vulnerability of labile residues to oxidation. em Tert /em -butyl hydroperoxide ( em t /em -BHP), hydrogen peroxide (H2O2), or 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) are often used as chemical stress reagents to evaluate protein oxidation [6,8,9,10,12,16,17,18]. In general, em t /em -BHP and H2O2 primarily oxidize Met residues via nucleophilic substitution reactions. In contrast, AAPH (a water-soluble radical initiator) oxidizes both Met and Trp residues [8,19] through free radical reactions. The azo compound, AAPH, is thermally unstable and can generate alkyl radicals at elevated temperatures. In the presence of oxygen, alkyl radicals can form peroxyl radicals [20], which can further form alkoxyl radicals [11]. These radical species can effectively oxidize Met, Trp, and other oxidation-labile residues in proteins. One advantage of using an azo-radical initiator, such as AAPH or azobisisobutyronitrile, as NU 6102 an oxidative reagent is that it can produce a controllable and reproducible amount of oxidizing species [19,21,22]. In previous work conducted by Ji et al. [9], researchers used oxidative reagents of em t /em -BHP, H2O2, and AAPH to study the mechanism of Met, Trp, and His oxidation in parathyroid hormone. Although Ji et al.s work provided valuable insights into the oxidation mechanisms and the corresponding stabilization strategy, the model protein, parathyroid hormone, used in the study is a small protein with minimal tertiary structure. On the other hand, oxidation in large proteins, such as mAbs, may not only cause oxidation of Met, Trp, His, or other labile residues, but also induce additional physicochemical degradations. For example, the previous work did not assess aggregation that can be observed during protein oxidation. Therefore, in this work, NU 6102 we evaluated the oxidative NU 6102 degradation of therapeutic mAbs under the AAPH stress, where we observed both mAb oxidation and aggregation. Protein aggregation is often induced by physical stresses, such as agitation [23], freeze/thawing, and freeze/drying processes [24]. The AAPH-induced aggregation observed here is likely created through covalent bonds as a result of free radical reactions. Protein aggregates can potentially trigger immune reactions and are considered as a critical quality attribute for restorative proteins [25]. Therefore, it is important to have a good understanding of the formation and the nature of protein aggregates associated with protein oxidation. In addition, this study also provides insight to reduce protein aggregation when exposing to oxidative reagents during restorative protein production and storage. 2. Results 2.1..In addition, we collected emission spectra of bityrosine standard to further support this hypothesis (Figure 5B). residues could account for the non-reducible cross-links. An excipient screening study shown that Trp, pyridoxine, or Tyr could efficiently reduce protein aggregation due to oxidative stress. This work provides important insight into the mechanisms of oxidative-stress induced protein aggregation, as well as strategies to minimize such aggregate formation during the development and storage of restorative proteins. strong class=”kwd-title” Keywords: monoclonal antibody, free radical, protein aggregation, oxidation, excipient 1. Intro Oxidation-induced degradation of restorative proteins is commonly observed during pharmaceutical developing, handling, and storage [1,2,3]. These oxidation reactions happen when there are triggered oxygen varieties including singlet oxygen (1O2), superoxide radical (O2??), hydroxyl radical (?OH), and peroxide (-OO-) in the environment. Reactive oxygen species can be produced through light, warmth, free radicals, or transition metals [4,5]. These varieties can oxidize methionine (Met), tryptophan (Trp), histidine Mouse monoclonal to EphA1 (His), tyrosine (Tyr), and cysteine (Cys) residues in proteins [6]. For example, it has been reported that ultraviolet (UV) irradiation induced oxidation of Met and Trp residues inside a monoclonal antibody (mAb) [7], thermal or chemical stresses caused Met oxidation in proteins [8,9,10], and free radicals advertised Tyr oxidation in ATPase [11]. Oxidative modifications can alter proteins secondary and tertiary constructions [12], hydrophobicity [13], stability [14], biological activity [14], and plasma blood circulation half-life [15]. Owing to these potential effects, oxidation is typically an important quality attribute to be closely monitored during the development of therapeutic proteins. Various stress models including light and chemical stresses are often used to determine potential oxidation sizzling spots in restorative proteins and to characterize the vulnerability of labile residues to oxidation. em Tert /em -butyl hydroperoxide ( em t /em -BHP), hydrogen peroxide (H2O2), or 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) NU 6102 are often used as chemical stress reagents to evaluate protein oxidation [6,8,9,10,12,16,17,18]. In general, em t /em -BHP and H2O2 primarily oxidize Met residues via nucleophilic substitution reactions. In contrast, AAPH (a water-soluble radical initiator) oxidizes both Met and Trp residues [8,19] through free radical reactions. The azo compound, AAPH, is definitely thermally unstable and may generate alkyl radicals at elevated temperatures. In the presence of oxygen, alkyl radicals can form peroxyl radicals [20], which can further form alkoxyl radicals [11]. These radical varieties can efficiently oxidize Met, Trp, and additional oxidation-labile residues in proteins. One advantage of using an azo-radical initiator, such as AAPH or azobisisobutyronitrile, as an oxidative reagent is definitely that it can produce a controllable and reproducible amount of oxidizing varieties [19,21,22]. In earlier work carried out by Ji et al. [9], experts used oxidative reagents of em t /em -BHP, H2O2, and AAPH to study the mechanism of Met, Trp, and His oxidation in parathyroid hormone. Although Ji et al.s work provided handy insights into the oxidation mechanisms and the corresponding stabilization strategy, the model protein, parathyroid hormone, used in the study is a small protein with minimal tertiary structure. On the other hand, oxidation in large proteins, such as mAbs, may not only cause oxidation of Met, Trp, His, or additional labile residues, but also induce additional physicochemical degradations. For example, the previous work did not assess aggregation that can be observed during protein oxidation. Therefore, with this work, we evaluated the oxidative degradation of restorative mAbs under the AAPH stress, where we observed both mAb oxidation and aggregation. Protein aggregation is often induced by physical tensions, such as agitation [23], freeze/thawing, and freeze/drying processes [24]. The AAPH-induced aggregation observed here is likely created through covalent bonds as a result of free radical reactions. Protein aggregates can potentially trigger immune reactions and are considered as a critical quality attribute for restorative proteins [25]. Therefore, it is important to have a good understanding of the formation and the nature of protein aggregates associated with.