Presenter Information
Kaitlyn Shaw, Gayathri Aparnasai Reddy, and Wilson S Meng
Graduate School of Pharmaceutical Sciences, School of Pharmacy, Duquesne University, Pittsburgh, PA 15282
Abstract
Purpose: Immunoglobulin gamma (IgG) is the primary structural platform for therapeutic monoclonal antibodies (mAbs). Highly concentrated (>100 mg/mL) mAb formulations are increasingly used to extend drug action but are susceptible to protein denaturation and irreversible aggregation due to physical and chemical stresses, including temperature and pH changes. Aggregation can reduce drug potency and increase immunogenicity, creating challenges for the quality and safety of biologic products. This study aimed to develop and validate a rapid, sensitive, and cost-effective method for detecting protein aggregation in IgG solutions.
Methods: Human plasma-derived IgG was reconstituted in phosphate-buffered saline (0.001–0.02 mg/mL) and exposed to heat stress (60°C for 15 minutes) to induce aggregation. Arginine was added to selected samples as an aggregation-suppressing control. Aggregates were quantified using an ELISA-based assay in polystyrene microtiter plates blocked with 2% bovine serum albumin (BSA). Following incubation, bound protein was detected using horseradish peroxidase-conjugated anti-human IgG and tetramethylbenzidine substrate. Absorbance was measured at 450 nm.
Results: Aggregated IgG exhibited increased affinity for the polystyrene surface and displaced weakly adsorbed BSA, whereas native monomeric IgG remained in solution. Reduced aggregation was observed in arginine-containing samples, supporting assay validity. The assay demonstrated a 24.9% increase in selectivity relative to the blank (1.25-fold increase). The limit of detection and limit of quantification were 0.094% and 0.285% (w/v), respectively.
Conclusion: This plate-based ELISA method provides a rapid, sensitive, and economical approach for monitoring IgG aggregation and may serve as a useful component of biologic quality-control programs.
Advisor
Wilson S Meng
Submission Type
Poster
Exploring a Facile ELISA-based Method for Detecting Antibody Aggregates
Purpose: Immunoglobulin gamma (IgG) is the primary structural platform for therapeutic monoclonal antibodies (mAbs). Highly concentrated (>100 mg/mL) mAb formulations are increasingly used to extend drug action but are susceptible to protein denaturation and irreversible aggregation due to physical and chemical stresses, including temperature and pH changes. Aggregation can reduce drug potency and increase immunogenicity, creating challenges for the quality and safety of biologic products. This study aimed to develop and validate a rapid, sensitive, and cost-effective method for detecting protein aggregation in IgG solutions.
Methods: Human plasma-derived IgG was reconstituted in phosphate-buffered saline (0.001–0.02 mg/mL) and exposed to heat stress (60°C for 15 minutes) to induce aggregation. Arginine was added to selected samples as an aggregation-suppressing control. Aggregates were quantified using an ELISA-based assay in polystyrene microtiter plates blocked with 2% bovine serum albumin (BSA). Following incubation, bound protein was detected using horseradish peroxidase-conjugated anti-human IgG and tetramethylbenzidine substrate. Absorbance was measured at 450 nm.
Results: Aggregated IgG exhibited increased affinity for the polystyrene surface and displaced weakly adsorbed BSA, whereas native monomeric IgG remained in solution. Reduced aggregation was observed in arginine-containing samples, supporting assay validity. The assay demonstrated a 24.9% increase in selectivity relative to the blank (1.25-fold increase). The limit of detection and limit of quantification were 0.094% and 0.285% (w/v), respectively.
Conclusion: This plate-based ELISA method provides a rapid, sensitive, and economical approach for monitoring IgG aggregation and may serve as a useful component of biologic quality-control programs.