The effect of mAb and excipient cryoconcentration on long-term frozen storage stability - Part 1: Higher molecular weight species and subvisible particle formation

Cryoconcentration upon large-scale freezing of monoclonal antibody (mAb) solutions leads to regions of different ratios of low molecular weight excipients, like buffer species or sugars, to protein. This study focused on the impact of the buffer species to mAb ratio on aggregate formation after frozen storage at -80 degrees C, -20 degrees C, and -10 degrees C after 6 weeks, 6 months, and 12 months. An optimised sample preparation was established to measure T-g ' of samples with different mAb to histidine ratios via differential scanning calorimetry (DSC). After storage higher molecular weight species (HMWS) and subvisible particles (SVPs) were detected using size-exclusion chromatography (SEC) and FlowCam, respectively. For all samples, sigmoidal curves in DSC thermograms allowed to precisely determine T-g ' in formulations without glass forming sugars. Storage below T-g ' did not lead to mAb aggregation. Above T-g ', at -20 degrees C and - 10 degrees C, small changes in mAb and buffer concentration markedly impacted stability. Samples with lower mAb concentration showed increased formation of HMWS. In contrast, higher concentrated samples led to more SVPs. A shift in the mAb to histidine ratio towards mAb significantly increased overall stability. Cryoconcentration upon large-scale freezing affects mAb stability, although relative changes compared to the initial concentration are small. Storage below T-g ' completely prevents mAb aggregation and particle formation.