Conformational implications of an inversed pH-dependent antibody aggregation

Area of Science:

• Biopharmaceutical development
• Protein aggregation kinetics
• Antibody stability

Background:

• Antibody formulation development utilizes accelerated stability studies at elevated temperatures.
• The pH- and temperature-dependence of antibody aggregation is complex.
• Understanding aggregation mechanisms is crucial for accurate shelf-life prediction.

Purpose of the Study:

• To investigate the pH- and temperature-dependence of aggregation for a human monoclonal IgG2 antibody.
• To explore the impact of different storage conditions on antibody stability and aggregation kinetics.
• To evaluate the validity of accelerated stability data for predicting antibody shelf-life.

Main Methods:

• Monitoring dimer and high molecular weight aggregate formation at various temperatures (4, 29, 37°C) and pH conditions.
• Analyzing aggregation kinetics using Arrhenius plots.
• Employing differential scanning calorimetry to assess thermal unfolding.
• Investigating aggregation of specific IgG2 structural isoforms.

Main Results:

• A human IgG2 antibody displayed typical pH-dependent dimer formation at normal temperatures but inverse pH-dependence for high molecular weight aggregates at 37°C.
• Nonlinear Arrhenius kinetics were observed across different storage conditions.
• Differential scanning calorimetry showed no thermal unfolding at or below 37°C.
• Clip-mediated aggregation at low pH and elevated temperatures was identified as a key factor in increased aggregate formation, influenced by IgG2 isoform conformational differences.

Conclusions:

• The complex, nonlinear Arrhenius kinetics challenge the reliability of accelerated stability data for predicting IgG2 antibody shelf-life.
• Conformational differences among IgG2 isoforms contribute to unique aggregation profiles, particularly clip-mediated aggregation at elevated temperatures and low pH.
• Further research into antibody aggregation mechanisms is necessary to refine stability assessment strategies.