Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmaceutical Science

Background:

• Monoclonal antibodies (mAbs) are crucial therapeutics.
• Accurate detection of mAb aggregates is vital for drug safety and efficacy.
• Current methods like SEC can be time-consuming and may miss certain aggregates.

Purpose of the Study:

• To explore extrinsic fluorescence as a high-throughput detection tool for monoclonal antibody aggregates.
• To evaluate the sensitivity and correlation of a fluorescent probe assay with SEC.
• To assess the potential of this method for analyzing protein aggregation.

Main Methods:

• Thermal stress was applied to several IgG molecules.
• High molecular weight species (aggregates) were separated using size-exclusion chromatography (SEC).
• Extrinsic fluorescence of SYPRO Orange was used to study isolated aggregates and monomers.

Main Results:

• SYPRO Orange showed high sensitivity to structurally altered IgG aggregates compared to native forms.
• The fluorescence assay demonstrated a strong correlation with SEC in quantifying IgG aggregates.
• The method showed potential for detecting protein particles not amenable to SEC analysis.

Conclusions:

• Extrinsic fluorescence with SYPRO Orange is a promising high-throughput tool for detecting IgG aggregates.
• This method can be applied to pharmaceutical solutions and for studying protein aggregation kinetics.
• The technique may aid in identifying early-stage protein particle formation.