| RRC ID |
65133
|
| 著者 |
Senga Y, Imamura H, Miyafusa T, Watanabe H, Honda S.
|
| タイトル |
AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.
|
| ジャーナル |
Sci Rep
|
| Abstract |
Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.
|
| 巻・号 |
7(1)
|
| ページ |
12466
|
| 公開日 |
2017-9-29
|
| DOI |
10.1038/s41598-017-12693-w
|
| PII |
10.1038/s41598-017-12693-w
|
| PMID |
28963557
|
| PMC |
PMC5622108
|
| MeSH |
Animals
CHO Cells
Cricetulus
Drug Stability
High-Throughput Screening Assays*
Hot Temperature
Humans
Hydrogen-Ion Concentration
Immunoglobulin Fc Fragments / chemistry*
Immunoglobulin G / chemistry*
Immunoglobulins, Intravenous / chemistry*
Kinetics
Peptides / chemical synthesis
Peptides / chemistry*
Protein Aggregates
Protein Binding
Quality Control
Solutions
|
| IF |
3.998
|
| リソース情報 |
| ヒト・動物細胞 |
293T(RCB2202) |
