Identification of IgG1 Aggregation Initiation Region by Hydrogen Deuterium Mass Spectrometry

Noda, Masanori; Ishii, Kentaro; Yamauchi, Mika; Oyama, Hiroaki; Tadokoro, Takashi; Maenaka, Katsumi; Torisu, Tetsuo; Uchiyama, Susumu

doi:10.1016/j.xphs.2019.02.023

Cited by 15 publications

(8 citation statements)

References 33 publications

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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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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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“…Binding regions between antibodies could be predicted by computational calculation 7 or directly investigated by hydrogen deuterium exchange mass spectrometry (HDX-MS). 8,9 However, it is still challenging to select amino acid residues to be replaced on an mAb sequence because even a single residue replacement impacts the physical properties of mAbs. 10 At the manufacturing and quality control stages, in addition to optimization of culture condition and purification process, a formulation development for mAbs has been attempted.…”

Section: Introductionmentioning

confidence: 99%

Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody

Oyama

Koga

Tadokoro

et al. 2020

Journal of Pharmaceutical Sciences

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“…For the induced-fit model, a little low cooperativity may be allowed only when the variable domains remain their fold enough to recognize antigen upon losing interdomain stability. According to the previous DSC analyses using intact IgGs, T m values derived from Fab domain for intact rituximab, adalimumab and pertuzumab are available to be 76 °C, 70 °C and 77 °C, respectively (15; 29; 23). Of the three antibodies, only pertuzumab scFv showed a single peak transition in DSC analysis, and the T m 2 is 69.7 °C, which differs by approximately 7 °C.…”

Section: Discussionmentioning

confidence: 99%

Thermostability and binding properties of single-chained Fv fragments derived from therapeutic antibodies

Tadokoro,

Tsuboi,

Nakamura

et al. 2024

Preprint

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Small antibody fragments have recently been used as alternatives to full-length monoclonal antibodies in therapeutic applications. One of the most popular fragment antibodies is single-chain fragment variables (scFvs), consisting of variable heavy (VH) and variable light (VL) domains linked by a flexible peptide linker. scFvs have small molecular sizes, which enables good tissue penetration and low immunogenicity. Despite these advantages, the use of scFvs, especially for therapeutic purpose, is still limited because of the difficulty to regulate the binding activity and conformational stability. In this study, we constructed and analyzed 10 scFv fragments derived from 10 representatives of FDA-approved mAbs to evaluate their physicochemical properties. Differential scanning calorimetry analysis showed that scFvs exhibited relatively high but varied thermostability, from 50 to 70 degrees of melting temperatures, and different unfolding cooperativity. Surface plasmon resonance analysis revealed that scFvs fragments that exhibit high stability and cooperative unfolding likely tend to maintain antigen binding. This study demonstrated the comprehensive physicochemical properties of scFvs derived from FDA-approved antibodies, providing insights into antibody design and development.

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Identification of IgG1 Aggregation Initiation Region by Hydrogen Deuterium Mass Spectrometry

Cited by 15 publications

References 33 publications

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

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

Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody

Thermostability and binding properties of single-chained Fv fragments derived from therapeutic antibodies

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