Impact of Initial Aggregate Level on Aggregation Potential of Monoclonal Antibodies in Different Buffer Systems.

Purpose: This study investigates the stability and kinetics of degradation when monoclonal antibodies (mAbs) process intermediates are stored in commonly used Protein A elution buffers, including citrate, acetate, and glycine, at varying pre-existing aggregates levels (low: 1-5%, moderate: 5-15% and high: 15-25%) at 4°C and 30°C to simulate standard and worst-case conditions.

Methodology: mAb samples were subjected to thermal stress to achieve different levels of initial aggregates. The pre-aggregated samples were then incubated in different buffers at 4°C and 30°C to assess aggregation rates and stability. Aggregates were quantified using dynamic light scattering (DLS) integrated with machine learning (ML).

Result: At 30°C, half-life reductions for citrate, acetate, and glycine buffers were 6.30-fold, 6.48-fold, and 9.64-fold, respectively, compared to 4°C, with glycine buffer offering the best stability, while citrate buffer provides the least. At higher initial aggregate levels, half-lives decreased by 2.15-, 1.95-, and 1.73-fold for citrate, acetate, and glycine buffers, respectively, compared to lower initial aggregates. Second-order kinetics dominated in samples having lower initial aggregate levels, while first-order kinetics prevailed in medium and high initial aggregate levels. Glycine buffer at 4°C with low initial aggregates achieved the highest half-life of 129 days, whereas citrate buffer at 30°C with high initial aggregate exhibited the lowest stability, with a half-life of 3.5 days.

Conclusion: The findings highlight the significance of using an optimal buffer system and appropriate storage conditions for in-process intermediates during mAb manufacturing to have a robust process that delivers safe and efficacious biotherapeutic products.