AlphaScreen-based Homogeneous Assay Using a Pair of 25-residue Artificial Proteins for High-Throughput Analysis of Non-Native IgG

Sci Rep. 2017 Sep 29;7(1):12466. doi: 10.1038/s41598-017-12693-w.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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


  • Immunoglobulin Fc Fragments
  • Immunoglobulin G
  • Immunoglobulins, Intravenous
  • Peptides
  • Protein Aggregates
  • Solutions