Variations in critical quality attributes (CQAs) on therapeutic monoclonal antibodies (mAbs) due to cultivation conditions, storage conditions and external stress-factors

Sammanfattning

Ensuring close control of critical quality attributes (CQAs), such as post-translational modifications (PTMs), in therapeutic monoclonal antibodies (mAbs) is essential for maintaining function and batch homogeneity in large-scale production. Common PTMs include deamidation, fragmentation, oxidation, charge variants, and glycosylations. This study aimed to evaluate CQA variations and correlate the findings to cultivation conditions, post-collection storage, and three external stress factors. Samples were collected after 7-14 days of cultivation from four batches, with specific conditions, and evaluated by orthogonal methods. Batch 1 (50 L) was stored in a freezer; batch 2 (1000 L), and batches 3 and 4 (5 L each) were stored in a fridge. Three stressors were applied: 1) high pH + 37°C, 2) hydrogen peroxide, and 3) mechanical stress. Analysis methods included Size Exclusion High-Performance Liquid Chromatography, Capillary Electrophoresis Sodium Dodecyl Sulfate, Capillary Isoelectric Focusing, Hydrophilic Interaction Liquid Chromatography, Liquid Chromatography Mass Spectrometry, and Backgrounded Membrane Imaging. Results indicated that earlier sample collection promoted better CQAs, suggesting earlier harvest. The study did not investigate mAb yield, necessary to weigh quality against product outcome. All stressors affected the CQAs differently depending on the stressor, and should all be avoided if possible. Batch 1, with 50 L cultivation volume, freezer storage, and cell separation directly after collection, showed the best retained CQAs and the most robust mAbs. The study concluded that batch 1's conditions were optimal, but further investigation including triplicates, statistical analysis, and consistent measurement points is required to confirm the conclusion.