Effect of UVC Irradiation on the Oxidation of Histidine in Monoclonal Antibodies

Miyahara, Yuki; Shintani, Koya; Hayashihara-Kakuhou, Kayoko; Zukawa, Takehiro; Morita, Yosuke; Nakazawa, Takashi; Yoshida, Takuya; Ohkubo, Tadayasu; Uchiyama, Susumu

doi:10.1038/s41598-020-63078-5

Cited by 25 publications

(22 citation statements)

References 32 publications

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“…For example, Amano et al reported no correlation between pKa of His residues and His oxidation, 82 whereas Miyahara et al reported His residues with lower pKa values were more prone to photooxidation. 88 The discrepancy in the reported role of pKa on His oxidation could be due to the different stress conditions (i.e., MCO versus UVC light exposure) used in these studies and the different by-products formed after the stress condition (i.e., oxo-His versus Asn and Asp residues). Compared to Met and Trp oxidation, in silico prediction of His oxidation is understudied.…”

Section: Oxidationmentioning

confidence: 86%

In silico prediction of post-translational modifications in therapeutic antibodies

Vatsa¹

2022

mAbs

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Monoclonal antibodies are susceptible to chemical and enzymatic modifications during manufacturing, storage, and shipping. Deamidation, isomerization, and oxidation can compromise the potency, efficacy, and safety of therapeutic antibodies. Recently, in silico tools have been used to identify liable residues and engineer antibodies with better chemical stability. Computational approaches for predicting deamidation, isomerization, oxidation, glycation, carbonylation, sulfation, and hydroxylation are reviewed here. Although liable motifs have been used to improve the chemical stability of antibodies, the accuracy of in silico predictions can be improved using machine learning and molecular dynamic simulations. In addition, there are opportunities to improve predictions for specific stress conditions, develop in silico prediction of novel modifications in antibodies, and predict the impact of modifications on physical stability and antigen-binding.

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Section: Oxidationmentioning

confidence: 86%

In silico prediction of post-translational modifications in therapeutic antibodies

Vatsa¹

2022

mAbs

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“…In our previous study, an in vitro analysis using a histidine analog N -benzoyl-histidine demonstrated that N -benzoyl-histidine was initially oxidized at the C-2 position of the imidazole ring to form N -benzoyl-2-oxo-histidine by a free radical-generating system (copper/ascorbate), and the formed N -benzoyl-2-oxo-histidine was subsequently oxidized to form a series of further oxidized products via the ring opening reaction of the imidazole ring [ 47 ]. The mono-oxigenized modification of histidine was also detected on various polypeptides, including copper/ascorbate-treated bovine serum albumin or β-amyloid peptide, and ultraviolet C-irradiated immunoglobulin gamma 1 [ 48 , 49 , 50 , 51 ], and a similar degradation pathway of the 2-oxo-histidine residue has been proposed [ 51 , 52 ]. Thus, it is possible that the commercial carnosine and anserine may contain contaminants other than 2-oxo-forms, which are not detected in the experimental conditions used in this study.…”

Section: Discussionmentioning

confidence: 99%

2-Oxo-Imidazole-Containing Dipeptides Play a Key Role in the Antioxidant Capacity of Imidazole-Containing Dipeptides

et al. 2021

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There is substantial evidence for the antioxidant functions of imidazole-containing dipeptides (IDPs), including carnosine and anserine, under physiological and pathological conditions in vivo. However, the detailed mechanism underlying the antioxidant functions is still poorly understood. Recently, we discovered the endogenous production of 2-oxo-imidazole-containing dipeptides (2-oxo-IDPs), such as 2-oxo-carnosine and 2-oxo-anserine, as novel derivatives of IDPs in mouse tissues and revealed that the antioxidant capacity of 2-oxo-carnosine was much greater than that of carnosine. However, the antioxidant capacity of 2-oxo-IDPs still remains unclear. In this study, we evaluated 2-oxo-carnosine and 2-oxo-anserine by multiple in vitro assays, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing/antioxidant power, and oxygen radical absorbance capacity assays in comparison with the corresponding IDPs, carnosine and anserine. All the assays employed herein demonstrated that 2-oxo-carnosine and 2-oxo-anserine exhibited a greater antioxidant capacity than that of the corresponding IDPs. Quantitative high-performance liquid chromatography tandem mass spectrometry revealed that commercial IDPs standards were contaminated with a certain amount of 2-oxo-IDPs, which was correlated with the antioxidant capacity. DPPH radical scavenging assay revealed that the elimination of contaminated 2-oxo-IDPs from the IDPs standards caused a significant decrease in the antioxidant capacity compared to the original IDPs standards. These results suggest that the main driver of the antioxidant capacity of IDPs is 2-oxo-IDPs; accordingly, the conversion of IDPs to 2-oxo-IDPs may be a critical step in the antioxidant functions.

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“…Mass spectrometry-based methods have been widely used for characterizing biopharmaceuticals. 21,22,23 Also in AAV, mass spectrometry methods have been recently applied for structural analysis of VPs. Intact mass spectrometry (intact MS) measurement combined with peptide mapping results revealed that the N-terminal methionine residue was cleaved and the next alanine residue was acetylated in VP1 and VP3, whereas there was no acetylation in VP2 for multiple AAV serotypes (AAV1, AAV2, AAV5, AAV7, AAV9, and AAVrh10).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry

et al. 2021

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Recombinant adeno-associated virus (rAAV) is a leading platform in human gene therapy.The AAV capsid is composed of three viral proteins (VPs): VP1, VP2, and VP3. To ensure the safety of AAV-based gene therapy products, the stoichiometry of VPs of AAV vector should be carefully monitored. Here, SDS-PAGE, capillary gel electrophoresis (CGE), and liquid chromatography-UV-mass spectrometry (LC-UV-MS) were performed to evaluate the VP components of AAV1, AAV2, and AAV6. Two types of VP3 related components, VP3 variant and VP3 fragment, were identified. The VP3 variant was the N-terminal shorter VP3 of which the translation started at M211, not at the conventional initiation codon, M203. The VP3 variant could be generated by leaky scanning of the first initiation codon of VP3. We also showed that the VP3 variant was identified in a minor peak prior to VP3 in CGE measurement. Meanwhile, the VP3 fragment was the C-terminal cleaved VP3 of which the sequence of VP3 ended at D590 or D626, indicating that cleavage occurred between D590 and P591, or D626 and G627. The cause of the cleavage of the DP or DG sequence was hydrolysis due to low pH of the mobile phase and high temperature of the column oven in the LC system which was necessary to clearly separate the peak of VPs. VP3 fragments detected only in LC-UV-MS in small amount account with less than 3% of total peak area, should be included in the quantification of VP3. Finally, the relationship of VP stoichiometry determined by the above three methods was discussed. From this study, we

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Effect of UVC Irradiation on the Oxidation of Histidine in Monoclonal Antibodies

Cited by 25 publications

References 32 publications

In silico prediction of post-translational modifications in therapeutic antibodies

In silico prediction of post-translational modifications in therapeutic antibodies

2-Oxo-Imidazole-Containing Dipeptides Play a Key Role in the Antioxidant Capacity of Imidazole-Containing Dipeptides

Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry

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