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Updated: Oct 14, 2025
Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System
Published on: May 22, 2020
Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells
Yukako Senga1, Motomichi Doi1, Masayoshi Onitsuka2
1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Insights
Researchers developed a new method to detect intracellular immunoglobulin G (IgG) aggregates in cells using a fluorescently labeled artificial protein. This technique helps understand antibody aggregation during production and improve therapeutic antibody quality.
Area of Science:
- Biotechnology
- Protein Chemistry
- Cell Biology
Background:
- Recombinant immunoglobulin G (IgG) aggregation is a common issue during production.
- The mechanisms of intracellular and extracellular IgG aggregation in cell culture are not well understood.
- Current methods for assessing IgG aggregates are limited.
Purpose of the Study:
- To develop a novel method for detecting intracellular IgG aggregates.
- To investigate the factors influencing intracellular IgG aggregation in Chinese hamster ovary (CHO) cells.
- To explore the relationship between intracellular and extracellular IgG aggregates.
Main Methods:
- An artificial protein, AF.2A1, was engineered to bind non-native IgG conformers and aggregates.
- Fluorescently labeled AF.2A1 was conjugated and transfected into antibody-producing CHO cells.
- Intracellular aggregates were visualized and quantified using microscopy.
Main Results:
- Intracellular IgG aggregates were successfully detected in CHO cells using fluorescent AF.2A1.
- The amount of intracellular aggregates varied based on cell culture additives.
- A moderate correlation was observed between intracellular and extracellular IgG aggregates, suggesting secretion.
Conclusions:
- The developed AF.2A1-based method enables the investigation of intracellular antibody aggregation.
- This approach can guide the optimization of therapeutic antibody production for improved yield and quality.
Abstract:
Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.
